Libraries, arrays and their uses for targeted affinity enhancement

ABSTRACT

The present disclosure relates to methods and materials for enhancing the binding affinity of an antibody by means of generating a library or an array of targeted amino acid changes (e.g., mutations) at one or more positions in an antibody variable domain. The present disclosure relates to libraries or arrays and their uses for enhancing antibody affinity. The present disclosure relates to methods and materials for mutagenesis, including for the generation of novel or improved antibody variable domains and libraries or arrays of mutant antibody variable domains or nucleic acids encoding such mutant or modified variable domains.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of International Patent ApplicationNo. PCT/US2008/088639, filed on Dec. 31, 2008; U.S. ProvisionalApplication No. 61/018,113, filed Dec. 31, 2007; U.S. ProvisionalApplication No. 61/018,105, filed Dec. 31, 2007; and U.S. ProvisionalApplication No. 61/018,101, filed Dec. 31, 2007, the disclosures ofwhich are herein incorporated by reference in their entirety.

FIELD

The present disclosure relates to libraries or arrays and their uses forenhancing the binding affinity of an antibody. Modified antibodyvariable domains obtained by novel targeted affinity enhancement methodsdemonstrate an increased binding affinity compared to the bindingaffinity exhibited by the unmodified (parent) variable domain. Thepresent disclosure also relates to novel combinations of degeneratecodons that code for an equal representation of one or morenon-redundant amino acid changes.

BACKGROUND

Affinity enhancement of a monoclonal antibody (beyond the ordinarynanomolar affinity which is typically achieved in an animal system) isdesirable when producing a therapeutic agent, regardless of how theantibody was originally generated (e.g., by transgenic mice, by phagedisplay, by yeast display, or by ordinary murine hybridoma methods).Extremely high affinity antibodies (e.g., a scFv or Fab) may beadvantageous if they can be administered with equivalent efficacy inmuch lower doses, thereby decreasing the cost of producing the drugand/or diminishing its adverse side-effects.

Although natural immunological systems typically yield antibodies ofnanomolar (10⁻⁹ M) affinity, greater affinities may be desirable.However, since astronomical numbers of different antibody combiningsites are possible, it has been difficult to design a method forchoosing a few key mutations in an antibody variable domain which mightlead to greater binding affinity, particularly in the absence ofreliable structural (e.g., x-ray crystallographic) data. Presenttechniques for enhancing the affinity of an antibody often requirescreening a large number of antibody variants and may introduceundesirable mutations outside of the antibody binding pocket.

SUMMARY

The present disclosure relates to methods and materials for enhancingthe binding affinity of an antibody by means of generating a library orarray of targeted amino acid changes (e.g., mutations) at one or morepositions in an antibody variable region to enhance affinity. For themethods, in some embodiments, antibody variable region sequences may bealigned according to a standard numbering system such as Kabat. Thepresent disclosure relates to libraries or arrays and their uses forenhancing antibody affinity. The present disclosure also relates tonovel combinations of degenerate codons which code for an equalrepresentation of one or more non-redundant amino acid changes.

Methods are disclosed which minimize the total number of amino acidchanges for enhancement of an antibody's affinity. Such methods may makea number of amino acid changes at an original amino acid position.Further, groups of positions on an antibody variable region comprising aheavy and/or light chain variable region may be selected for change byemploying novel methods which assign each amino acid on the variableregion of the heavy and/or light chains of antibodies to one of thefollowing unique groups: contacting (C), peripheral (P), supporting (S),interfacial (I), or distant (D). These novel proximity groups permit theselection of amino acid residues that are candidates for change.Additionally or alternatively, positions for amino acid changes may bebased upon a novel method of determining the degree to which theoriginal amino acid residue differs from the corresponding consensus orgermline residue in terms of charge, size or chemical functionality. Forexample, the methods provided by the disclosure may include utilizationof tables of numerical components, which can be added together toidentify “conspicuous” amino-acid changes.

Methods are also disclosed for enhancing the affinity of a variableregion of an antibody (e.g., a heavy chain and/or light chain variableregion) by identifying the proximity assigned to amino acid positions inthe variable region of the antibody using the “prox” line as shown inFIG. 3A, 3B, 3C and/or 3D and preferably changing one or more contacting(C), supporting (S), peripheral (P) and/or interfacial (I) amino acidresidues, with other amino acids residues. Less preferably one or moredistant (D) amino acid residues may additionally or alternatively bechanged.

An exemplary method for affinity enhancement of an antibody variableregion (e.g., a heavy chain and/or light chain variable region) includesaligning a variable region sequence with consensus or individuallight-chain and heavy-chain sequences according to a standard numberingsystem such as Kabat; additionally or alternatively co-aligning with theantibody's own direct germline precursor sequences if they are known andpreferably changing one or more contacting (C), supporting (S),peripheral (P) and/or interfacial (I) amino acid residues, with otheramino acids residues. Less preferably one or more distant (D) amino acidresidues may additionally or alternatively be changed.

Methods are provided for enhancing the binding affinity of a variabledomain (e.g., a heavy chain and/or light chain variable region) of anantibody, to obtain a modified variable domain with enhanced bindingaffinity by using the “prox” line as shown in FIG. 3A, 3B, 3C and/or 3D;identifying the proximity assigned to amino acid positions in thevariable domain of the antibody as contacting (C), peripheral (P),supporting (S), interfacial (I) or distant (D); changing one or morecontacting (C), supporting (S), peripheral (P) interfacial (I) and/ordistant (D) amino acid residues, with other amino acids residues withother amino acid residues, thereby generating a library or array ofmodified variable domains; screening the library or array for bindingaffinity to a binding partner; and obtaining a modified variable domainwith enhanced binding affinity to the binding partner.

In some embodiments, the other amino acid residues are alanine (Ala, A),arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D),glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine(His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K),phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine(Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) or valine (Val, V).

In some embodiments, the other amino acid changes can be introduced bymutagenesis (e.g., PCR-based, Dpn1-based or Kunkel mutagenesis) usingprimers. Exemplary primers may comprise degenerate codons, including,for example, 2 to 12-fold degenerate codons. In preferred embodiments,the degenerate codons do not encode for cysteine or methionine. In someembodiments, basic amino acid changes may be introduced using thedegenerate codon ARG (R=A/G), which codes for arginine/lysine. In otherembodiments, polar amino acid changes may be introduced using thedegenerate codons WMC (W=A/T; M=A/C), which codes forserine/threonine/asparagine/tyrosine and/or CAS (S=C/G), which codes forhistidine/glutamine. In other embodiments, acidic amino acid changes maybe introduced using the degenerate codon GAS (S=C/G), which codes forglutamic acid/aspartic acid. In other embodiments, non-polar changes maybe introduced using the degenerate codons NTC (N=A/G/C/T), which codesfor leucine/phenylalanine/isoleucine/valine, KGG (K=G/T), which codesfor tryptophan/glycine and/or SCG (S=C/G), which codes forproline/alanine. An exemplary 7 primer set includes ARG, WMC, CAS, GAS,NTC, KGG and SCG which collectively encode eighteen amino acidsexcluding cysteine and methionine. Alternate degenerate codons can beutilized to produce eighteen amino acids. For example, in the example ofdegenerate codons given above, ARG can be replaced with ARA, WMC can bereplaced with WMT, CAS can be replaced with CAK (K=G,T), CAM (M=A or C),or CAW (W=A or T), NTC with NTT, SCG with SCA, SCC, or SCT. In addition,the single primer listed as NTC or NTT can be replaced with: two primersMTC, KTC (or MTT/KTT; MTC/KTT; MTT/KTC); STC, WTC (or STT/WTT; STT/WTC;STC/WTT); RTC, YTC (or RTT/YTT; RTC/YTT, RTT/YTC).

In some embodiments, degenerate codons may include, for example, NHT orNHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG orDGG (where B=C/G/T, D=A/G/T). An exemplary three primer set includesNHT, VAA and BGG which collectively encode eighteen amino acidsexcluding cysteine and methionine. Alternate degenerate codons can beutilized to produce eighteen amino acids and there are multiple codonsets that can be utilized. Either NHT or NHC (where N=A/G/C/T, H=A/C/T)can be utilized in combination with either VAG or VAA (where V=A/C/G)and either BGG or DGG (where B=C/G/T, D=A/G/T). In addition, the NHTprimer can be broken up into a multitude of different degenerate primersets. N can be broken up into B (CGT)+A; D (AGT)+C; H (ACT)+G; V(ACG)+T; K+M; S+W; R+Y; K+A+C; M+G+T; S+A+T; W+C+G; R+C+T; Y+G+A;A+C+G+T. For the second and third positions in the codon, the HT or HCwould continue to be utilized. If the first codon remains either N; K+M;S+W; or R+Y, then, H can be further broken down into A+Y; C+W; T+M; orA+C+T. The third position in the codon would remain T or C.

Methods are also provided for making a modified variable domain (e.g., aheavy chain and/or light chain variable region) of an antibody withenhanced binding affinity compared to a parent variable domain bymodifying the nucleotide sequence of an antibody variable domain atamino acid residues that encode preferably one or more contacting (C),peripheral (P), supporting (S), and/or interfacial (I) amino acidresidues identified from the “prox” line as shown in FIGS. 3A, 3B, 3Cand/or 3D to produce amino acid changes at the position, therebygenerating a library of modified antibody variable domains; andselecting a modified variable domain from the library that has enhancedbinding affinity. Less preferably one or more distant (D) amino acidresidues may additionally or alternatively be changed according todisclosed methods.

In some embodiments, the method further comprises contacting a parentvariable domain with a binding partner under conditions that permitbinding; contacting modified variable domain(s) with the binding partnerunder conditions that permit binding; and determining binding affinityof the modified variable domain(s) and the parent variable domain forthe binding partner, wherein modified variable domain(s) that have abinding affinity for the binding partner greater than the bindingaffinity of the parent variable domain for the binding partner areidentified as having enhanced binding affinity for the binding partner.

Methods are also provided for selecting a modified variable domain(e.g., a heavy chain and/or light chain variable region) of an antibodywith enhanced binding affinity to a binding partner compared to a parentvariable domain by obtaining a library of modified antibody variabledomains comprising amino acid changes at preferably multiple (e.g., 2,4, 6, 8, 10, 12, 14, 16, 18) contacting (C), peripheral (P), supporting(S), and/or interfacial (I) amino acid residues identified from the“prox” line as shown in FIG. 3A, 3B, 3C and/or 3D; determining thebinding affinity of the modified antibody variable domains and theparent variable domain to the binding partner; and selecting themodified antibody variable domains that have enhanced binding affinityto the binding partner compared to the parent variable domain. Forexample, basic amino acid changes can be introduced (e.g., arginine(Arg, R) and/or lysine (Lys, K), polar amino acid changes can beintroduced (e.g., serine (Ser, S), threonine (Thr, T), asparagine (Asn,N), tyrosine (Tyr, Y), histidine (His, H) and/or glutamine (Gln, Q)),acidic amino acid changes can be introduced (e.g., glutamic acid (Glu,E), and/or aspartic acid (Asp, D)), and/or non-polar amino acids can beintroduced (e.g., leucine (Leu, L), phenylalanine (Phe, F), isoleucine(Ile, I), valine (Val, V), tryptophan (Trp, W), glycine (Gly, G),proline (Pro, P) and/or alanine (Ala, A)).

Methods are also provided for enhancing the binding affinity of avariable domain (e.g., a heavy chain and/or light chain variable region)of an antibody, to obtain a modified variable domain with enhancedbinding affinity by using the “prox” line as shown in FIG. 3A, 3B, 3Cand/or 3D; identifying the proximity assigned to amino acid positions inthe variable domain of the antibody as contacting (C), peripheral (P),supporting (S), interfacial (I) or distant (D); preferably changing oneor more contacting (C) amino acid residues with other amino acidresidues, thereby generating a library or array of modified variabledomains; screening the library or array for binding affinity to abinding partner; and obtaining a modified variable domain with enhancedbinding affinity to the binding partner.

Methods are provided for producing a nucleic acid library with an equalrepresentation of one or more non-redundant amino acid changes at eachof one or more positions (e.g., contacting (C), peripheral (P),supporting (S), interfacial (I) or distant (D) positions) in a parentnucleic acid by providing a set of primers (e.g., 3, 7 or 9 primers)that each comprise at least one degenerate codon (e.g., 2 to 12-folddegenerate) at identical positions, wherein the primers arecomplementary to a sequence in the parent nucleic acid and the primerscode for an equal representation of non-redundant amino acid changes atone or more positions; hybridizing a primer from the set to the parentnucleic acid; amplifying the parent nucleic acid molecule with theprimer to generate one or more nucleic acids that code for amino acidchanges at one or more identical positions; repeating the hybridizationand amplification steps with remaining primers from the set; pooling thenucleic acids produced with each primer; and obtaining a library ofnucleic acids coding for an equal representation of one or more aminoacid changes at one or more identical positions, with the proviso thatthe degenerate codons do not code for methionine or cysteine.

A set of primers is provided that comprise at least one degenerate codonat identical positions (e.g., contacting (C), peripheral (P), supporting(S), interfacial (I) or distant (D) positions), wherein the degeneratecodons code for an equal representation of one or more non-redundantamino acid changes at each of one or more positions in the parentnucleic acid and the primers are complementary to a sequence in theparent nucleic acid, with the proviso that the degenerate codons do notcode for methionine or cysteine.

A kit is also provided for mutagenesis of one or more positions in aparent nucleic acid (e.g., contacting (C), peripheral (P), supporting(S), interfacial (I) or distant (D) positions), a set of primerscomprising at least one degenerate codon at identical positions, whereinthe degenerate codons code for an equal representation of one or morenon-redundant amino acid changes at each of one or more positions in theparent nucleic acid and the primers are complementary to a sequence inthe parent nucleic acid, with the proviso that the degenerate codons donot code for methionine or cysteine.

In some embodiments, the primer set codes for eighteen amino acidchanges at each of one or more positions in the parent nucleic acid. Insome embodiments, the set of primers each comprise a degenerate codonwhich collectively code for alanine, arginine, asparagine, asparticacid, glutamine, glutamine acid, glycine, histidine, isoleucine,leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine and valine at each position. In some embodiments, the set ofprimers comprises three primers. In some embodiments, the primers eachcomprise one or more degenerate codons as represented by NHT or NHC(where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG(where B=C/G/T, D=A/G/T). In some embodiments, the set of primerscomprises seven primers. In some embodiments, the primers each compriseone or more degenerate codons as represented by ARG (where R=A/G), WMC(where W=A/T and M=A/C), CAS (where S=C/G), GAS (where S=C/G), NTC(where N=A/G/C/T), KGG (where K=G/T) and SCG (where S=C/G).

In some embodiments, the primer set codes for basic amino acid changesat each of one or more positions in the parent nucleic acid. In someembodiments, the primer set comprises one primer. In some embodiment,the one primer comprises a degenerate codon which codes for arginine andlysine. In some embodiments, the one primer comprises one or moredegenerate codons as represented by ARG (where, R=A/G).

In some embodiments, the primer set codes for polar amino acid changesat each of one or more positions in the parent nucleic acid. In someembodiments, the primer set comprises two primers. In some embodiments,the two primers each comprise a degenerate codon which collectively codefor serine, threonine, asparagine and tyrosine. In some embodiments, thetwo primers each comprise one or more degenerate codons as representedby WMC (where, W=A/T; M=A/C) and CAS (where S=C/G).

In some embodiments, the primer set codes for acidic amino acid changesat each of one or more positions in the parent nucleic acid. In someembodiments, the primer set comprises one degenerate codon. In someembodiments, the one primer comprises a degenerate codon that codes forglutamic acid and aspartic acid. In some embodiments, the one primercomprises one or more degenerate codons as represented by GAS (whereS=C/G).

In some embodiments, the primers code for non-polar amino acid changesat each of one or more positions in the parent nucleic acid. In someembodiments, the primer set comprises three degenerate codons. In someembodiments, the three primers each comprise a degenerate codon thatcollectively code for glutamic acid and aspartic acid. In someembodiments, the primers each comprise one or more degenerate codons asrepresented by NTC (where, N=A/G/C/T), KGG (where, K=G/T), and SCG(where S=C/G).

In some embodiments, the parent nucleic acid encodes an antibodyvariable region. In some embodiments, the positions in the parentnucleic acid code for contacting (C), supporting (S), interfacial (I),peripheral (P) or distant (D) residues.

In some embodiments, the contacting (C) residue may be incomplementarity determining domain-1 (CDR1) in a light chain variabledomain. In certain embodiments, the contacting (C) residue may be at aposition corresponding to position 28, 30 or 31 in CDR1. In otherembodiments, the contacting (C) residue may be in CDR2 in a light chainvariable domain. In certain embodiments, the contacting (C) residue maybe at a position corresponding to position 50, 51 or 53 in CDR2. Inother embodiments, the contacting (C) may be in CDR3 in a light chainvariable region. In some embodiments, the contacting (C) residue may bein CDR1 in a heavy chain variable domain. In certain embodiments, thecontacting (C) residue may be at a position corresponding to position 32or 33 in CDR1. In some embodiments, the (C) contacting residue may be inCDR2 in a heavy chain variable domain. In certain embodiments, thecontacting (C) residue may be at a position corresponding to position50, 52, 53, 54, 56, or 58 in CDR2. In some embodiments, the contacting(C) may be in CDR3 in a heavy chain variable region.

In some embodiments, the methods further comprise inserting the modifiedantibody variable domain into an appropriate vector. In someembodiments, the vector is a plasmid, phage or phagemid. In certainembodiments, the vector is pXOMA Fab or pXOMA-gIII-Fab (see, e.g., FIG.6). The pXOMA Fab vector is similar to the pXOMA-gIII-Fab vector butdoes not have a pIII coding sequence.

In some embodiments, the variable domain is from a chimeric antibody. Inother embodiments, the variable domain is from a humanized or humanengineered antibody. In some embodiments, the variable domain is from ahuman antibody.

In some embodiments, binding affinity of a modified variable domain orparent variable domain to a binding partner is determined by measuringK_(off). In some embodiments, binding affinity of a modified variabledomain or parent variable domain to a binding partner may be measured byBiacore (e.g., Biacore 2000 or A100).

The present disclosure also provides method of mutagenesis of a parentnucleic acid encoding an antibody variable domain to generate modifiedantibody variable domains by obtaining one or more primers that eachcomprise at least one 2 to 12 fold degenerate codon, wherein each primercomprises at least two oligonucleotide sequences that are complementaryto a sequence in the parent nucleic acid and code for an amino acidmutation with the exception of cysteine or methionine at one amino acidposition encoded by the parent nucleic acid; and mutating the parentnucleic acid by replication or polymerase based amplification using theone or more obtained primers, wherein replication or amplification ofthe parent nucleic acid with the one or more primers generates mutatednucleic acids that encode modified antibody variable domains.

The present disclosure also provides methods for mutagenesis of anantibody variable domain to obtain modified antibody variable domainswith mutated amino acid sequences by identifying one or more amino acidpositions in the antibody variable domain for mutagenesis; substitutingone or more of the identified amino acid residues in the antibodyvariable domain with other amino acid residues excluding cysteine andmethionine to generate a library or an array of modified antibodyvariable domains with mutated amino acid sequences; screening thelibrary or array of modified antibody variable domains in an assay for abiological activity of the antibody variable domain; and obtainingmodified antibody variable domains having the biological activity of theantibody variable domain.

The present disclosure also provides for generating an array of nucleicacids encoding modified antibody variable domains by obtaining acollection of nucleic acids encoding modified antibody variable domainscontaining amino acid mutations other than cysteine and methionine atamino acid residues of a parent antibody variable domain sequence bymutagenesis of a nucleic acid encoding the antibody variable domainsequence using primers that each comprise at least one 2 to 12 folddegenerate codon; sequencing the collection of nucleic acids encodingthe modified antibody variable domains; and arranging each sequencednucleic acid encoding a modified antibody variable domain to generate anarray of nucleic acid sequences each encoding a modified antibodyvariable domain.

The present disclosure also provides methods for generating an array ofnucleic acid sequences encoding modified antibody variable domains bypreparing a plurality of nucleic acid sequences by mutagenesis thatencode a plurality of modified antibody variable domains that vary froma parent antibody variable domain sequence at one or more amino acidpositions and contain one of eighteen different amino acids excludingcysteine and methionine at each position mutated from the parent proteinsequence; and arranging each nucleic acid sequence prepared in step (a)to generate an array of nucleic acid sequences each encoding a modifiedantibody variable domain.

The present disclosure also provides methods for generating an array ofclones comprising nucleic acids encoding modified antibody variabledomains by preparing a plurality of nucleic acids by mutagenesis thatencode a plurality of modified antibody variable domains that vary froma parent antibody variable domain sequence at one or more amino acidpositions and contain one of eighteen different amino acids excludingcysteine and methionine at each position varied from the parent antibodyvariable domain sequence; transfecting the prepared nucleic acids intohost cells and selecting clones comprising the transfected nucleicacids; and arranging each selected clone to generate an array of cloneswith each arrayed clone capable of expressing a modified antibodyvariable domain.

The present disclosure also provides methods of producing a nucleic acidlibrary with an equal representation of non-redundant amino acid changesat an amino acid position encoded by a parent nucleic acid encoding anantibody variable domain by providing a set of primers that eachcomprise at least one degenerate codon, wherein each primer comprises atleast two oligonucleotide sequence that are complementary to a sequencein the parent nucleic acid and code for an amino acid mutation with theexception of cysteine and methionine at one amino acid position encodedby the parent nucleic acid, wherein the primers code for an equalrepresentation of non-redundant amino acid changes at the one position;hybridizing a primer from the set to the parent nucleic acid;replicating or amplifying the parent nucleic acid molecule with theprimer to generate nucleic acids that code for amino acid changes at theone position, repeating the hybridizing and replicating steps with eachremaining primer from the set; pooling the nucleic acids produced witheach primer; and obtaining a library of nucleic acids coding for anequal representation of amino acid changes at the one position.

The present disclosure also provides methods for obtaining a nucleicacid sequence with an improvement in comparison to a parent nucleic acidsequence encoding an antibody variable domain with respect to at leastone molecular or biological property of interest, said methodcomprising; obtaining a set of primers that each comprise at least one 2to 12 fold degenerate codon that does not code for cysteine andmethionine, wherein the primers are complementary to a sequence in theparent nucleic acid sequence and wherein the primers code fornon-redundant amino acid mutations at one amino acid position encoded bythe parent nucleic acid sequence; mutating the parent nucleic acidsequence by replication or polymerase based amplification using theobtained set of primers to generate variant nucleic acid sequences;producing a library or array of variant nucleic acid sequences codingfor amino acid mutations at the one position in the parent nucleic acidsequence; and screening the library or array of variant nucleic acidsequences to identify nucleic acid sequences that have a desirableimprovement in comparison with the parent nucleic acid sequence withrespect to at least one molecular or biological property of interest.

The present disclosure also provides methods of making modified antibodyvariable domains with mutated amino acid sequences by modifying theamino acid sequence of an antibody variable domain to produce amino acidmutations at an amino acid residue in the antibody variable domain togenerate a library or an array of modified antibody variable domainswith mutated amino acid sequences, wherein the amino acid mutationsexclude cysteine and methionine; and selecting modified antibodyvariable domains from the library or the array that have a biologicalactivity of an unmodified antibody variable domain.

The present disclosure also provides methods for selecting modifiedantibody variable domains with mutated amino acid sequences by obtaininga library or an array of modified antibody variable domains comprisingamino acid mutations at one amino acid residues in an amino acidsequence of a protein, wherein the amino acid mutations exclude cysteineand methionine; assaying the modified antibody variable domains for abiological activity of an unmodified protein; and selecting the modifiedantibody variable domains that have a biological activity of theunmodified antibody variable domain.

In some embodiments, the amino acid mutations are selected from thegroup consisting of: alanine, arginine, asparagine, aspartic acid,glutamine, glutamine acid, glycine, histidine, isoleucine, leucine,lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosineand valine at each position.

In some embodiments, the set of primers or primers code for eighteenamino acid mutations at the one amino acid position encoded by theparent nucleic acid.

In some embodiments, three primers that each comprise at least one 2 to12 fold degenerate codon are obtained or used. In some embodiments,seven primers that each comprise at least one 2 to 12 fold degeneratecodon are obtained or used. In some embodiments, the degenerate codonsare selected from the group consisting of: NHT or NHC (where N=A/G/C/T,H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/G/T,D=A/G/T). In some embodiments, the degenerate codons are selected fromthe group consisting of: ARG (where R=A/G), WMC (where W=A/T and M=A/C),CAS (where S=C/G), GAS (where S=C/G), NTC (where N=A/G/C/T), KGG (whereK=G/T) and SCG (where S=C/G).

In some embodiments, the primers code for basic amino acid mutations atthe one amino acid position encoded by the parent nucleic acid. In someembodiments, one primer that comprises at least one 2 to 12 folddegenerate codon is obtained. In some embodiments, the one primercomprises a degenerate codon which codes for arginine and lysine. Insome embodiments, the degenerate codon is represented by ARG (where,R=A/G).

In some embodiments, the primers code for polar amino acid mutations atthe one amino acid position encoded by the parent nucleic acid. In someembodiments, two primers that comprise at least one 2 to 12 folddegenerate codon is obtained. In some embodiments, the two primerscomprise degenerate codons that collectively code for serine, threonine,asparagine and tyrosine. In some embodiments, the degenerate codons arerepresented by WMC (where, W=A/T; M=A/C) and CAS (where S=C/G).

In some embodiments, the primers code for acidic amino acid mutations atthe one amino acid position encoded by the parent nucleic acid. In someembodiments, one primer that comprises at least one 2 to 12 folddegenerate codon is obtained. In some embodiments, the one primercomprises a degenerate codon that codes for glutamic acid and asparticacid. In some embodiments, the degenerate codon is represented by GAS(where S=C/G).

In some embodiments, the primers code for non-polar amino acid mutationsat the one amino acid position encoded by the parent nucleic acid. Insome embodiments, three primers that comprise at least one 2 to 12 folddegenerate codon are obtained. In some embodiments, the three primerscomprise degenerate codons that collectively code for glutamic acid andaspartic acid. In some embodiments, the degenerate codons arerepresented by NTC (where, N=A/G/C/T), KGG (where, K=G/T), and SCG(where S=C/G).

In some embodiments, the methods may further comprising selecting theone or more positions in the parent nucleic acid sequence for mutation.

In some embodiments, the position for mutation encodes one or more CDRresidues. In some embodiments, the CDRs are defined by Kabat, Chothia orIMGT. In some embodiments, all CDR resides are mutated.

In some embodiments, modified antibody variable domains are selectedthat have increased activity as compared to the unmodified antibodyvariable domain. In some embodiments, modified antibody variable domainsare selected that have decreased activity as compared to the unmodifiedantibody variable domain. In some embodiments, modified antibodyvariable domains are selected that have equal activity as compared tothe unmodified antibody variable domain.

In some embodiments, the mutagenesis or substitution is performed withone or more primers that each comprise at least one 2 to 12 folddegenerate codon, wherein each primer comprises at least twooligonucleotide sequences that are complementary to a sequence in aparent nucleic acid and code for an amino acid substitution with theexception of cysteine and methionine at one amino acid position encodedby the parent nucleic acid.

The present disclosure also provides a library or an array comprisingvariants of a antibody variable domain sequence, wherein the variantseach comprise an amino acid mutation at one amino acid position in thesequence of a parent antibody variable domain and wherein the amino acidmutations are not cysteine or methionine.

The present disclosure also provides methods for obtaining a nucleicacid sequence with an improvement in comparison to a parent nucleic acidsequence encoding an antibody variable domain with respect to at leastone molecular or biological property of interest by mutating the parentnucleic acid by polymerase based amplification using one or more primersthat each comprise at least one 2 to 12 fold degenerate codon togenerate mutated nucleic acid sequences, wherein each primer comprisesat least two oligonucleotide sequences that are complementary to asequence in the parent nucleic acid and code for an amino acid mutationwith the exception of cysteine or methionine at one amino acid positionencoded by the parent nucleic acid; sequencing the mutated nucleic acidsequences; arranging each sequenced mutated nucleic acid sequencecomprising one amino acid mutation to generate an array of mutatednucleic acid sequences; and screening the array of variant nucleic acidsequences to identify nucleic acid sequences that have a desirableimprovement in comparison with the parent nucleic acid sequence withrespect to at least one molecular or biological property of interest.

In some embodiments, modified antibody variable domains are selectedthat have increased activity as compared to the unmodified protein. Insome embodiments, modified antibody variable domains are selected thathave decreased activity as compared to the unmodified protein. In someembodiments, modified antibody variable domains are selected that haveequal activity as compared to the unmodified protein.

The present disclosure also provides antibodies or binding fragmentsthereof made by the methods of the present disclosure.

Additional features and advantages are described herein, and will beapparent from, the following Detailed Description and the Figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a generalized schematic map of an exemplary antibody combiningsite as described herein, looking downward onto the “top” surface of avariable domain comprising a light chain variable region and a heavychain variable region. It shows the six CDR loops (L1, L2, L3, H1, H2,H3) which are spatially located directly above the three-dimensionalstructure of the evolutionarily-conserved framework underneath. As shownand discussed herein, this map provides roughly approximate higher-orderstructural information, which is not available from the linear primarysequence alone, such as the identity of potential nearest neighbors inthe space-filling model of a generic variable domain. Specific featuresof the murine ING1 monoclonal antibody have been added to this map, sothat it can also call attention to localized domains of the antibody'scombining site containing clusters of high-conspicuousness positions asdescribed herein, which are likely to be in contact with sidechains onthe antigen. In particular, each amino-acid position in the murine ING1antibody is represented on this map by a white rectangle containing agroup of symbols. The letter and number at the bottom-left of eachrectangle (e.g., “H 98” in CDR-loop H3) is the Kabat-position number ofthe amino-acid residue on the antibody molecule within either chain(L=light, H=heavy). The small upper-case letter (e.g., “B”) at thebottom-right is a designation for the residue's proximity as describedherein (C=Contacting, P=Peripheral, S=Supporting, I=Interfacial)relative to the antibody's binding site (shown on the “prox” line inFIGS. 2A-2D). The large upper-case letter (e.g., “A”) at the upper-leftis the amino-acid code for the residue's sidechain (line “murING1” inFIGS. 2A-2D). The large single digit at the upper right (e.g., “3”) insome rectangles is the non-zero conspicuousness-value as describedherein of affinity enhancement for the sidechain (line “cspc” in FIGS.2A-2D), calculated in reference to the appropriate human consensussequence for light chain (hK2) or heavy chain (hH 1). Rectangles with nosuch value reflect a conspicuousness of zero.

FIGS. 2A-2D: Alignments of sequences in the light chain and heavy chain,with lines (e.g., prox, cspc) relating to affinity enhancement and linesrelating to human engineering (e.g., risk) are shown. In each set oflines, the top ones apply the present disclosure to the murine ING1antibody (2A-2B), and the bottom ones relate the present disclosure tothe general principles of human engineering (Studnicka et al., ProteinEngineering, 7(6):805-814 (1994); U.S. Pat. No. 5,766,886). Each set oflines shows the Kabat position numbers (pos), the general classificationof proximity groups for each position of every antibody (prox), themurine ING1 monoclonal antibody sequence to be affinity-enhanced(murING1), the conspicuousness value as described herein of eachposition for affinity-enhancement when the murine ING1 antibody iscompared to murine consensus sequences (cspc), several murine consensussequences to which ING1 is compared (mK2 or mH2a), the human ING1residues which are introduced during the HUMAN ENGINEERING™ process(humING1), the degree of disconnection of the sidechain from theantibody's combining site (disc) as described herein, the degree ofoutward-orientation of the sidechain on the antibody's surface (outw) asdescribed herein, the degree of risk for human engineering (risk), andthe Kabat position numbers (pos) (2A-2B). Similarly, FIGS. 2C and 2D arealignments of sequences in the light chain and heavy chain of IL-1antibody (also referred to as cA5 and/or XPA23), with lines (e.g., prox,cspc) relating to affinity enhancement and lines relating to humanengineering (e.g., risk).

FIGS. 3A-3D are mutual alignments of consensus sequences (Kabat et al.(1991) (eds), Sequences of Proteins of Immunological Interest, 5th ed.)for major murine and human subgroups of the light chain and heavy chain.Each alignment relates them to the proximity groups as described hereinfor each position (prox), and the Kabat position numbers (pos).

FIG. 4 shows a chart of the numerical components which can be addedtogether to calculate each amino acid's affinity-enhancementconspicuousness value, including the components for changes inclass-and-charge, for changes in physical size due to somatic mutation,and for repeated identical mutations at the same position in multiplehomologous antibodies.

FIG. 5 shows PCR mutagenesis of CDR3 utilizing the CDR-H3oligonucleotide H3-3NP2 (SEQ ID NO: 267): 5′-GCTACATATTTCTGTGCAAGATTTGGCTCTKGGGTGGACTACTGGGGTCAAGG-3′, which introduces an amino acidsubstitution into CDR3, and the reverse primer Notl-R (SEQ ID NO: 285):5′-AGCGGCCGCACAAGATTTGGGCTCAACTCTC-3′, which incorporates the Notlrestriction site into the PCR product.

FIG. 6 depicts the plasmid map of the pXOMA-gIII-Fab vector. The vectoris 5,202 base pairs in length and has Ascl and Notl restriction sitesflanking the heavy chain encoding sequences, and HindIII and Asclrestriction sites flanking the light chain encoding sequences. The heavychain encoding sequences are fused to pIII encoding sequences in thevector. The pXOMA-Fab vector is similar but lacks the pIII encodingsequences.

FIG. 7 depicts the strategy for creating the light chain combinationvariants.

FIG. 8 depicts the strategy for creating the heavy chain combinationvariants.

FIG. 9A-9B shows CDR1, CDR2 and CDR3 as identified by the Kabat, Chothiaand IMGT numbering scheme for ING-1 (9A) and XPA23 (9B).

FIG. 10A-10D depict a continuous numbering scheme for the heavy andlight chain of XPA23 (10A and 10B, respectively). Consecutive numberingfrom position 1 in the light chain continues in the heavy chain suchthat position 1 in the heavy chain is also assigned number 108 since thelight chain sequence ends at number 107. Boxed residues indicate CDRsidentified by the IMGT method. FIGS. 10C and 10D show a continuousnumbering scheme for the heavy and light chain of ING-1 (10C and 10D,respectively).

FIG. 11: Periplasmic extracts of clones containing one of the eighteenpreferred amino acid mutations at Heavy Chain contacting positions inING-1 were tested on Biacore for improved off-rate (see example 7).Clones with greater than 1.9-fold decrease in off-rate are listed.

FIG. 12: Periplasmic extracts of clones containing one of the eighteenpreferred amino acid mutations at Light Chain contacting positions inING-1 were tested on Biacore for improved off-rate (see example 7).Clones with greater than 1.9-fold decrease in off-rate are listed.

FIG. 13: Periplasmic extracts of clones containing one of the eighteenpreferred amino acid mutations at Heavy Chain contacting positions inXPA23 were tested on Biacore for improved off-rate (see example 7).Clones with greater than 1.9-fold decrease in off-rate are listed.

FIG. 14: Periplasmic extracts of clones containing one of the eighteenpreferred amino acid mutations at Light Chain contacting positions inXPA23 were tested on Biacore for improved off-rate (see example 7).Clones with greater than 1.9-fold decrease in off-rate are listed.

FIG. 15A-15D depicts two modified IgGs with an A102F or 102Gsubstitution that were prepared and evaluated by Biacore with improvedaffinity (15B-15C, respectively) as compared to the parental (15A) ING-1antibody. 15D shows the affinity determination kinetics for both themodified and parental ING-1 antibodies.

FIG. 16A-16C are sensogram profiles depicting ING-1 light chain bindingto Ep-Cam.

FIG. 17 depicts modified ING-1 antibodies each comprising two or moreheavy chain mutations as compared to the parental antibody. Combinationsof heavy chain mutations yield affinity improvements up to 25-fold overthe parental ING-1 antibody. Affinity improvements are driven largely byimprovements in k_(off).

FIG. 18 shows amino acid substitutions at position 32 in the light chainvariable region of XPA23. Generally the substitutions at position 30decreased kd of the antibody-antigen interaction compared to theparental antibody.

FIG. 19 shows amino acid substitutions at position 30 in the light chainvariable region of XPA23. Generally the substitutions at position 30resulted in a comparable kd of the antibody-antigen interaction comparedto the parental antibody.

FIG. 20 shows amino acid substitutions at position 45 in the heavy chainvariable region of XPA23. Generally the substitutions improved kd of theantibody-antigen interaction at this position compared to the parentalantibody.

DETAILED DESCRIPTION

The present disclosure provides methods for enhancing the bindingaffinity of an antibody by means of generating a library or array oftargeted amino acid changes (e.g., mutations) at one or more positionsin an antibody variable domain. These methods for targeted affinityenhancement may be utilized even in the complete absence of any detailedinformation about the interaction between the antibody and its bindingpartner. The methods of the present disclosure do not require anythree-dimensional x-ray crystallographic structures of the chosenantibody's combining site with its binding partner and/or any type ofenergy-minimization algorithm. Such targeted amino acid changes at oneor more positions in an antibody variable domain that result in enhancedbinding as compared to a parent variable domain may be combined in asingle antibody variable domain. As used herein, array refers to anordered arrangement of members, including, for example, clones,periplasmic extracts, cell lysates, polynucleotides or nucleic acids andpolypeptides or proteins.

The present disclosure also provides methods for enhancing the affinityof an variable region of an antibody by identifying the proximityassigned to one or more amino acid positions in the variable domain ofthe antibody using the “prox” line as shown in FIG. 3A, 3B, 3C and/or 3Dand preferably changing one or more contacting (C), supporting (S),peripheral (P) and/or interfacial (I) amino acid residues, with otheramino acids residues. Less preferably one or more distant (D) amino acidresidues may additionally or alternatively be changed.

In an exemplary method to accomplish targeted affinity enhancement,amino acid residues may be selected for change by aligning a light chainor heavy chain variable region sequence of an antibody and comparing thesequence with any other variable region sequence (e.g., a homologousconsensus sequence for the light and heavy chain subgroups to which itis most similar, and/or with its own precursor germline sequence if itis available). Using the sequence alignment and the “prox” line shown inFIG. 3A, 3B, 3C and/or 3D to identify the proximity assigned to aminoacid positions in the variable region of a light chain and/or heavychain as contacting (C), peripheral (P), supporting (S), interfacial (I)and/or distant (D), amino acid residues may be selected for change.

Additionally or alternatively, the primary amino-acid sequence may becharacterized to identify amino acid residues that are “conspicuous”(e.g., by calculations as described herein) and that may be candidatesfor change. Residues differing markedly in charge or size or chemicalfunctionality from the corresponding residues in the selected sequence,including, for example, the consensus or the germline, may conferspecific affinity for antigen upon the antibody.

Amino acid positions identified as preferably contacting (C), peripheral(P), supporting (S) and/or interfacial (I) may be changed to other aminoacid residues to create a library or array of modified antibody variabledomains. Less preferably one or more distant (D) amino acid residues mayadditionally or alternatively be changed. Selected amino acid residuesmay be changed with other naturally occurring and/or synthetic aminoacid residues to create a library or multiple libraries and/or an arrayor multiple arrays of modified variable domains.

Modified variable domains may have one or more amino acid changes atpreferably one or more contacting (C), peripheral (P), supporting (S)and/or interfacial (I) amino acid residues identified from the “prox”line as shown in FIG. 3A, 3B, 3C and/or 3D which provides for enhancedbinding affinity. Less preferably one or more distant (D) amino acidresidues may additionally or alternatively be changed. The library orarray of modified antibody variable domains may be screened to identifythose modified antibody variable domains that bind to a binding partnerwith increased affinity as compared to the unmodified (parent) variabledomain.

The present disclosure also provides methods for producing a nucleicacid library or array with an equal representation of one or morenon-redundant amino acid changes at each of one or more positions in aparent nucleic acid. Such methods may be used to introduce classes(e.g., polar, non-polar, basic and acidic) of amino acid changes at oneor more positions in a parent nucleic acid. The methods may be used tointroduce eighteen amino acid changes (e.g., alanine, arginine,asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine and valine) at one or more positions in a parentnucleic acid by using a set of primers comprising between three and nineprimers each with a degenerate codon at an identical position. Certainamino acids may be excluded from the primer set (e.g., cysteine andmethionine). Further, a set of non-redundant degenerate codons (e.g.,collectively coding for eighteen codons) permits an over-representationof substitutions at each position compared to libraries of the same sizecreated using redundant degenerate codons (e.g., degenerate codons thatindividually or collectively code for thirty-two or sixty-four codons).This over-representation of amino acid substitutions results in asmaller library size with greater diversity. Without being bound by atheory of the invention, it is hypothesized that the use of the abovedegenerate codons can allow evaluation of how side chain functionalitiesaffect the binding interaction with the target at the positions ofinterest (e.g., contacting positions, etc.). For example, the use of theARG codon can probe the effect of a positive charge upon the affinity ofthe antibody towards the target. Similarly the GAS codon can probe theeffect of a negative charge, the WMC and CAS codons a polarsubstitution, and the NTC, KGG and SCG codons a non-polar substitution.

Methods for producing a nucleic acid library or array with an equalrepresentation of eighteen non-redundant amino acid changes at each ofone or more contacting (C), peripheral (P), supporting (S), interfacial(I) or distant (D) positions in a parent nucleic acid encoding anantibody variable domain may comprise providing a set of three primersthat each comprise one or more degenerate codons as represented by NHTor NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG orDGG (where B=C/G/T, D=A/G/T), wherein the primers are complementary to asequence in the parent nucleic acid and the primers code for an equalrepresentation of non-redundant amino acid changes at one or morepositions; hybridizing a primer from the set to the parent nucleic acid;amplifying the parent nucleic acid molecule with the primer to generateone or more nucleic acids that code for amino acid changes at one ormore identical positions; repeating the hybridization and amplificationsteps with remaining primers from the set; pooling the nucleic acidsproduced with each primer; and obtaining a library or array of nucleicacids coding for an equal representation of eighteen amino acid changesat one or more identical positions, with the proviso that the degeneratecodons do not code for methionine or cysteine.

Methods for producing a nucleic acid library or array with an equalrepresentation of eighteen non-redundant amino acid changes at each ofone or more contacting (C), peripheral (P), supporting (S), interfacial(I) or distant (D) positions in a parent nucleic acid encoding anantibody variable domain may comprise providing a set of seven primersthat each comprise one or more degenerate codons as represented by ARG(where R=A/G), WMC (where W=A/T and M=A/C), CAS (where S=C/G), GAS(where S=C/G), NTC (where N=A/G/C/T), KGG (where K=G/T) and SCG (whereS=C/G), wherein the primers are complementary to a sequence in theparent nucleic acid and the primers code for an equal representation ofnon-redundant amino acid changes at one or more positions; hybridizing aprimer from the set to the parent nucleic acid; amplifying the parentnucleic acid molecule with the primer to generate one or more nucleicacids that code for amino acid changes at one or more identicalpositions; repeating the hybridization and amplification steps withremaining primers from the set; pooling the nucleic acids produced witheach primer; and obtaining a library or array of nucleic acids codingfor an equal representation of eighteen amino acid changes at one ormore identical positions, with the proviso that the degenerate codons donot code for methionine or cysteine.

Methods for producing a nucleic acid library or array with an equalrepresentation of non-redundant basic amino acid changes at each of oneor more contacting (C), peripheral (P), supporting (S), interfacial (I)or distant (D) positions in a parent nucleic acid encoding an antibodyvariable domain may comprise providing a set of one primer thatcomprises one or more degenerate codons as represented by ARG (where,R=A/G), wherein the primer is complementary to a sequence in the parentnucleic acid and the primer codes for an equal representation ofnon-redundant amino acid changes at one or more positions; hybridizing aprimer from the set to the parent nucleic acid; amplifying the parentnucleic acid molecule with the primer to generate one or more nucleicacids that code for amino acid changes at one or more identicalpositions; and obtaining a library or array of nucleic acids coding foran equal representation of basic amino acid changes at one or moreidentical positions, with the proviso that the degenerate codons do notcode for methionine or cysteine.

Methods for producing a nucleic acid library or array with an equalrepresentation of non-redundant acidic amino acid changes at each of oneor more contacting (C), peripheral (P), supporting (S), interfacial (I)or distant (D) positions in a parent nucleic acid encoding an antibodyvariable domain may comprise providing a set of one primer thatcomprises one or more degenerate codons as represented by GAS (whereS=C/G), wherein the primer is complementary to a sequence in the parentnucleic acid and the primer codes for an equal representation ofnon-redundant amino acid changes at one or more positions; hybridizing aprimer from the set to the parent nucleic acid; amplifying the parentnucleic acid molecule with the primer to generate one or more nucleicacids that code for amino acid changes at one or more identicalpositions; and obtaining a library or array of nucleic acids coding foran equal representation of acidic amino acid changes at one or moreidentical positions, with the proviso that the degenerate codons do notcode for methionine or cysteine.

Methods for producing a nucleic acid library or array with an equalrepresentation of non-redundant polar amino acid changes at each of oneor more contacting (C), peripheral (P), supporting (S), interfacial (I)or distant (D) positions in a parent nucleic acid encoding an antibodyvariable domain may comprise providing a set of two primers that eachcomprise one or more degenerate codons as represented by WMC (where,W=A/T; M=A/C) and CAS (where S=C/G), wherein the primers arecomplementary to a sequence in the parent nucleic acid and the primerscode for an equal representation of non-redundant amino acid changes atone or more positions; hybridizing a primer from the set to the parentnucleic acid; amplifying the parent nucleic acid molecule with theprimer to generate one or more nucleic acids that code for amino acidchanges at one or more identical positions; repeating the hybridizationand amplification steps with remaining primers from the set; pooling thenucleic acids produced with each primer; and obtaining a library orarray of nucleic acids coding for an equal representation of polar aminoacid changes at one or more identical positions, with the proviso thatthe degenerate codons do not code for methionine or cysteine.

Methods for producing a nucleic acid library or array with an equalrepresentation of non-redundant non-polar amino acid changes at each ofone or contacting (C), peripheral (P), supporting (S), interfacial (I)or distant (D) more positions in a parent nucleic acid encoding anantibody variable domain may comprise providing a set of three primersthat each comprise one or more degenerate codons as represented by NTC(where, N=A/G/C/T), KGG (where, K=G/T), and SCG (where S=C/G), whereinthe primers are complementary to a sequence in the parent nucleic acidand the primers code for an equal representation of non-redundant aminoacid changes at one or more positions; hybridizing a primer from the setto the parent nucleic acid; amplifying the parent nucleic acid moleculewith the primer to generate one or more nucleic acids that code foramino acid changes at one or more identical positions; repeating thehybridization and amplification steps with remaining primers from theset; pooling the nucleic acids produced with each primer; and obtaininga library or array of nucleic acids coding for an equal representationof non-polar amino acid changes at one or more identical positions, withthe proviso that the degenerate codons do not code for methionine orcysteine.

The present disclosure also provides an ING-1 heavy chain variableregion as set forth in SEQ ID NO: 579 that comprises a substitution atposition 28 or 30 in HCDR1. In some embodiments, the substitution atposition 28 is selected from the group consisting of: T28V, T281 andT28P. In some embodiments, the substitution at position 30 is T30Y.

The present disclosure also provides an ING-1 heavy chain variableregion as set forth in SEQ ID NO: 579 that comprises a substitution atposition 59 in HCDR2. In some embodiments, the substitution at position59 is T59W.

The present disclosure also provides an ING-1 heavy chain variableregion as set forth in SEQ ID NO: 579 that comprises a substitution atposition 100, 101 or 102 in HCDR3. In some embodiments, the substitutionat position 100 is G100R. In some embodiments, the substitution atposition 101 is selected from the group consisting of: S101K, S101Q,S101V, S101I, S101G. In some embodiments, the substitution at position102 in HCDR3 is selected from the group consisting of: A102R, A102H,A102Y, A102W, A102F and A102G.

The present disclosure also provides an ING-1 light chain variableregion as set forth in SEQ ID NO: 580 that comprises a substitution atposition 28 or 29 in LCDR1. In some embodiments, the substitution atposition 28 in LCDR1 is selected from the group consisting of: S28R,S28K, S28H, S28Y, S28F, S28Q, S28V, S28I and S28L. In some embodiments,the substitution at position 29 in LCDR1 is selected from the groupconsisting of L29S and L29A.

The present disclosure also provides an ING-1 light chain variableregion as set forth in SEQ ID NO: 580 that comprises a substitution at54, 55 or 58 in LCDR2. In some embodiments, the substitution at position54 in LCDR2 is selected from the group consisting of: Y54K and Y54L. Insome embodiments, the substitution at position 55 in LCDR2 is selectedfrom the group consisting of: Q55R, Q55H and Q55W. In some embodiments,the substitution at position 58 in LCDR2 is selected from the groupconsisting of: N58W, N58V, N58I and N58P.

The present disclosure also provides an ING-1 light chain variableregion as set forth in SEQ ID NO: 580 that comprises a substitution atposition 97, 98, 99 or 100 in LCDR3. In some embodiments, thesubstitution at position 97 in LCDR3 is L97I. In some embodiments, thesubstitution at position 98 in LCDR3 is selected from the groupconsisting of: E98R, E98K, E98T, E98S and E98L. In some embodiments, thesubstitution at position 99 in LCDR3 is L99I. In some embodiments, thesubstitution at position 100 in LCDR3 is P100Y.

The present disclosure also provides an ING-1 antibody that comprises aheavy chain variable region as set forth in SEQ ID NO: 579 and a lightchain variable region as set forth in SEQ ID NO: 580, wherein the heavychain variable region and/or light chain variable region comprise one ormore of the substitutions in HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and/orLCDR3 as disclosed herein.

The present disclosure also provides an XPA-23 light chain variableregion as set forth in SEQ ID NO: 582 that comprises a substitution atposition 27, 28 or 29 in LCDR1. In some embodiments, substitution atposition 27 in LCDR1 is selected from the group consisting of: Q27S,Q27F and Q27G. In some embodiments, the substitution at position 28 inLCDR1 is selected from the group consisting of: D281, D28S and D28W. Insome embodiments, the substitution at position 30 in LCDR1 is N30F.

The present disclosure also provides an XPA-23 light chain variableregion as set forth in SEQ ID NO: 582 that comprises a substitution atposition 51 or 53 in LCDR2. In some embodiments, the substitution atposition 51 in LCDR2 is A51G. In some embodiments, the substitution atposition 53 in LCDR2 is selected from the group consisting of: S53K andS53R.

The present disclosure also provides an XPA-23 light chain variableregion as set forth in SEQ ID NO: 581 that comprises a substitution atposition 92, 93, 95 or 96 in LCDR3. In some embodiments, thesubstitution at position 92 in LCDR3 is D92S. In some embodiments, thesubstitution at position 93 in LCDR3 is selected from the groupconsisting of: S93D and S93E. In some embodiments, the substitution atposition 95 in LCDR3 is selected from the group consisting of: P95S andP95A. In some embodiments, the substitution at position 96 in LCDR3 isL96W.

The present disclosure also provides an XPA-23 heavy chain variableregion as set forth in SEQ ID NO: 581 that comprises a substitution atposition 135, 138, 139, 140 or 142 in HCDR1. In some embodiments, thesubstitution at position 135 in HCDR1 is selected from the groupconsisting of: T135K and T135E. In some embodiments, the substitution atposition 138 in HCDR1 is selected from the group consisting of: K138Y,K138W, K138E, K138L, K138P and K138H. In some embodiments, thesubstitution at position 139 in HCDR1 is Y139H. In some embodiments, thesubstitution at position 140 in HCDR1 is F1401. In some embodiments, thesubstitution at position 142 in HCDR1 is selected from the groupconsisting of: F142T and F142A.

The present disclosure also provides an XPA-23 heavy chain variableregion as set forth in SEQ ID NO: 581 that comprises a substitution atposition 161 or 163 in HCDR2. In some embodiments, the substitution atposition 161 in HCDR2 is selected from the group consisting of: S161Rand S161K. In some embodiments, the substitution at position 163 inHCDR2 is selected from the group consisting of: G163L, G163Q, G163W,G163Y, G163I, G163K, G163R and G163F.

The present disclosure also provides an XPA-23 heavy chain variableregion as set forth in SEQ ID NO: 581 that comprises a substitution atposition 208, 210, 211 or 212 in HCDR3. In some embodiments, thesubstitution at position 208 in HCDR3 is Y208L. In some embodiments, thesubstitution at position 210 in HCDR3 is G210V. In some embodiments, thesubstitution at position 211 in HCDR3 is selected from the groupconsisting of: N211A and N211V. In some embodiments, the substitution atposition 212 in HCDR3 is selected from the group consisting of: S212Eand S212P.

The present disclosure also provides an XPA-23 antibody that comprises aheavy chain variable region as set forth in SEQ ID NO: 581 and a lightchain variable region as set forth in SEQ ID NO: 582, wherein the heavychain variable region and/or light chain variable region comprise one ormore of the substitutions in HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and/orLCDR3 as disclosed herein.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentdisclosure, exemplary methods and materials are described.

Characterization of Amino Acid Residues in an Antibody Variable Domain

The present disclosure provides novel methods to assign each amino acidin an antibody heavy and/or light chain variable region to one of thefollowing unique groups, which includes, contacting (C), peripheral (P),supporting (S), interfacial (I), or distant (D) residues, as shown, forexample, on the “prox” lines of FIG. 2A, 2B, 2C, 2D, 3A, 3B, 3C and/or3D. For example, each of the more-than-200 amino-acid positions in anantibody's variable light chain and heavy chain has been designated as amember of one of these five novel groups. The “prox” line as shown inFIG. 3A, 3B, 3C and/or 3D is useful for any variable region sequence,irrespective of the specific amino acid sequence, such that residues canbe selected as candidates for change (e.g., any and/or all contacting(C) residues). Additionally or alternatively, methods are provided thatidentify the presence of conspicuous amino-acid residues which may becandidates for change. Conspicuous amino acid changes may differ incharge or size or chemical functionality from the corresponding residuesin the selected sequence (e.g., consensus or germline sequence) andrepresent positions where amino acid changes may enhance affinity.

Complementarity determining regions (CDRs) in an antibody variabledomain (e.g., LCDR1, LCDR2 and LCDR3 for the light chain and HCDR1,HCDR2 and HCDR3 for the heavy chain) may be defined according to anyknown method in the art including, for example, Kabat, Chothia or IMGT.Kabat, Chothia and IMGT definitions of CDRs 1-3 in the light chain andthe heavy chain of ING-1 and XPA23 is shown in FIGS. 9A and 9B,respectively. According to Kabat, LCDR1 comprises amino acid residues 24to 34, LCDR2 comprises amino acid residues 50 to 56, LCDR3 comprisesamino acid residues 89 to 97, HCDR1 comprises amino acid residues 31 to35b, HCDR2 comprises amino acid residues 50 to 65 and HCDR3 comprisesamino acid residues 95 to 102. According to Chothia, LCDR1 comprisesamino acid residues 24 to 34, LCDR2 comprises amino acid residues 50 to56, LCDR3 comprises amino acid residues 89 to 97, HCDR1 comprises aminoacid residues 26 to 32, HCDR2 comprises amino acid residues 52 to 56 andHCDR3 comprises amino acid residues 95 to 102. According to IMGT, LCDR1comprises amino acid residues 27 to 32, LCDR2 comprises amino acidresidues 50 to 52, LCDR3 comprises amino acid residues 89 to 97, HCDR1comprises amino acid residues 26 to 33, HCDR2 comprises amino acidresidues 51 to 57 and HCDR3 comprises amino acid residues 93 to 102.Residues numbers for the Kabat, Chothia and IMGT CDRs are given as Kabatposition numbers.

Exemplary methods for characterization of amino acid residues in anantibody binding domain may include: a determination of each amino acidresidue's proximity group as designated on the “prox” line of FIG. 2A,2B, 2C, 2D, 3A, 3B, 3C and/or 3D and additionally or alternatively adetermination of each amino acid residue's conspicuousness as calculatedby the methods provided in the present disclosure.

A. Determination of Proximity Groups

The characterization process may determine the proximity group for eachamino-acid position simply by inspecting the corresponding symbol(“CPSI.:”) on the “prox” lines as shown, for example, in FIG. 2A, 2B, 2Cand/or 2D. In some embodiments, the antibody's light-chain and/orheavy-chain sequences are aligned with appropriate sequences (e.g., suchas consensus or germline sequences) and also with the “prox” lines ofthe present methods (FIGS. 2A, 2B, 2C and/or 2D),

Each position in the light chain and heavy chain has been assigned toone of five novel groups designated as contacting (C), peripheral (P),supporting (S), interfacial (I), or distant (D) on the “prox” lines, forexample, of FIG. 2A, 2B, 2C, 2D, 3A, 3B, 3C and/or 3D according to themethods disclosed herein. These Figures (e.g., 2A, 2B, 2C, 2D, 3A, 3B,3C and/or 3D) contain a disc line to reflect disconnection from anysignificant effect upon an antibody's binding site, and an outw line toreflect outward-orientation on an antibody's surface.

Table 1 shows five proximity groups, as well as a novel designation ofdisconnection (as shown on a “disc” line, for example, in FIG. 2A, 2B,2C, 2D, 3A, 3B, 3C and/or 3D) and outward-orientation (shown as an“outw” line, for example, in FIG. 2A, 2B, 2C, 2D, 3A, 3B, 3C and/or 3D)as defined for each group. The number of positions of each type ofproximity group for an exemplary antibody (e.g., ING-1, as describedherein) in a light chain, a heavy chain, and both chains together areshown in Table 2.

TABLE 1 Proximity Abbr Disc/Outw Contacting C −+ −o Peripheral P o+ ooSupporting S −− o− Interfacial I −= o= += Distant • ++ +o +− p c

TABLE 2 Proximity L H L + H Contacting 16 21 37 Peripheral 3 8 11Supporting 14 16 30 Interfacial 9 10 19 Distant 70 63 133

Without being bound by a theory of the invention, it has beenhypothesized that amino acid residues designated as contacting (C) arelocated within the combining site (see, e.g., “−” on the “disc” line ofFIG. 2A, 2B, 2C and/or 2D), and their sidechains are mostlyoutward-oriented (see, e.g., “+” or “o” on the outw line). It has beenfurther hypothesized that these are generally surface-exposed residuesin the CDR loops themselves, so their sidechains are very favorablysituated for making direct contact with corresponding residues on abinding partner.

Without being bound by a theory of the invention, it has beenhypothesized that amino acid residues designated as peripheral (P) areslightly disconnected from the binding site (see, e.g., “o” on the“disc” line), and their sidechains are mostly outward-oriented (see,e.g., “+” or “o” on the outw line). Many of these are framework residueswith variable orientation, which are located at curves or twists in thepolypeptide chain not too far from CDR loops. Although they may normallynot make direct contact with a binding partner, they may possibly makecontact if a particular binding partner is bound preferentially towardone side of the binding site instead of being centered.

Without being bound by a theory of the invention, it has beenhypothesized that amino acid residues designated as supporting (S) areeither directly within or close to the combining site (see, e.g., “−” or“o” on the “disc” line), and their sidechains are inward-oriented, e.g.,“−” on the outw line). It has been further hypothesized that many ofthese residues are buried in the Vernier-zone platform directlyunderneath a combining site, so that their nonpolar sidechains are ableto act as conformation-stabilizing “anchors” for CDR loops which rest ontop of them.

Without being bound by a theory of the invention, it has beenhypothesized that amino acid residues designated as interfacial (I) maybe located anywhere in relation to the binding site (see, e.g., “+” or“o” or “−” on the “disc” line), but their sidechains form the interfacebetween the light and heavy subunits of the variable domain (see, e.g.,“_” on the outw line). It has been further hypothesized that amino acidchanges of these residues may cause the two subunits to pivot or rotaterelative to one another along their shared hydrophobic interfacialsurface, producing strong allosteric effects upon an entire bindingsite, for example, all six CDR loops may be forced to change theirconformation in response.

Without being bound by a theory of the invention, it has beenhypothesized that amino acid residues designated as distant (D) are oftwo different types, with those of the first type being disconnectedfrom a combining site and its targeted epitope (see, e.g., “+” on the“disc” line), and their sidechains may have any orientation exceptinterfacial (see, e.g., “+” or “o” or “−” but not “=” on the outw line).It is further hypothesized that amino acid changes at these positionsgenerally will have little or no effect on enhanced affinity to abinding partner.

B. Determination of Conspicuousness

In some embodiments, alternatively or additionally with determination ofthe proximity groups by inspection of the “prox” lines, thecharacterization process may involve a calculation of theconspicuousness value for each amino-acid position. The conspicuousnessvalue of a sidechain at a particular antibody position is hypothesizedto represent the degree to which it appears strikingly different orunusually outstanding in comparison with selected sequences (e.g., aconsensus or germline sequence). Without being bound by a theory of theinvention, this value indicates the likelihood that this particularresidue may be a somatic mutation which was necessary to confer bindingpartner specific affinity upon an antibody. Consequently, theconspicuousness value also correlates with the hypothesis that a newengineered amino acid substitution at or near this position couldpossibly lead to forming or strengthening a bond with a residue on abinding partner surface.

Conspicuousness values are calculated by comparing each sidechain of acandidate antibody with the corresponding sidechain of an appropriateconsensus or germline sequence, for example, from a mutual alignment.For example, numerical values for conspicuousness can be calculatedreadily for each amino-acid position in a given antibody, according tothe following formula: add 1 point for each three units of difference insize (e.g., divide the absolute value of the size-difference by 3 anddrop the decimal without rounding); add 1 point for a shift from onesidechain class to another; add 1 point for each unit (absolute value)of difference in charge, and add 1 point for nonidentity (see, e.g.,FIG. 4).

For example, where a single antibody sequence is aligned or comparedwith a single consensus or germline sequence, there is one “pair” ofsequences being compared. The conspicuousness value for each amino-acidposition in the alignment or comparison is the sum of the points forchemical function and physical size and nonidentity at that position.Where more than two sequences are aligned or compared together at thesame time, each of the antibody sequences may form a separate “pair”with each of the consensus or germline sequences. The conspicuousnessvalues are calculated as described (e.g., sum of function and size andnonidentity) for each pair of sequences being aligned or compared, andthen the overall conspicuousness value for each amino acid position inthe whole alignment is the sum of the values obtained from each pair atthat position, while also adding in a value for repeated identicalmutations.

It is hypothesized that nonidentity simply marks an amino-acid positionas minimally conspicuous if it displays any kind of difference whencompared with a corresponding consensus or germline position. Even aconservative mutation (e.g., from leucine to isoleucine or valine) maysuggest a possible bond with a binding partner, especially if a slightchange of size or shape was necessary to fine-tune steric relationshipsbetween the two molecules.

An exemplary calculation of conspicuousness is illustrated as follows.Four monoclonal antibodies to the same epitope were isolated, andportions of their heavy chains were mutually aligned with a germlinesequence, between Kabat positions 25 and 57 [Mendez et al., NatureGenetics, 15:146-152 (1997)] (see, Table 3). Since this alignmentcontains more than two sequences, each of the four antibody sequencescan separately form a “pair” with the one germline sequence. Thus,conspicuousness values are calculated separately for each of the fourpairs, and then totaled at each amino-acid position, while also addingin the additional values for repeated identical mutations.

TABLE 3 prox: PSSCSCCCCSISI.I.:...I.ISSCSCCCCCC pos:    30        40        50 germ: GSISSGGYYWSWIRQHPGKGLEWIGYIYYSGST mAb1:   N  D                  S     N mAb2:       D   T                    NmAb3:   v   D        p         HL    N mAb4:    N  D              DC

Three repetitions are shown in Table 3, at positions 28 and 31 and 56.In each of these cases, an identical amino acid (N or D) has appeared atthe same location in more than one independently isolated antibody.Accordingly, as described herein, these positions are given very highconspicuousness in the affinity enhancement process. An additional 2points are added for each repetition of an identical amino acid at agiven position (e.g., four D's amount to three repetitions of the firstD, so it is worth 3×2=6 points).

In an example, at position 50, the first pair (germ:mAb 1) gets 3 points(Y to S=2 for size+0 for class+0 for charge+1 for nonidentity), thesecond pair (germ:mAb2) gets 0 points (unmutated Y=0+0+0+0), the thirdpair (germ:mAb3) gets 3 points (Y to H=0 for size+1 for class+1 forcharge+1 for nonidentity), and the fourth pair (germ:mAb4) gets 0 points(unmutated Y=0+0+0+0). The total conspicuousness for position 50 is thesum (3+0+3+0) of these, plus 0 extra points for no repeated identicalmutations, which finally gives 6.

In another example, at position 28, the first pair gets 1 point (S toN=0+0+0+1), the second and third pairs get 0 points, and the fourth pairgets I point. Since the somatic mutation N appears at position 28 twice,it is repeated once, and thus gets 2 extra points. The totalconspicuousness for position 28 is the sum (1+0+0+1), plus 2 points forone repetition, which finally gives 4.

In another example, at position 31, each of the four pairs gets 4 points(G to D=1+1+1+1). Since the somatic mutation D appears at position 31four times, it is repeated three times, and thus gets 3×2=6 extrapoints. The total conspicuousness for position 28 is the sum (4+4+4+4),plus 6 points for three repetitions, which finally gives 22.

The conspicuousness points can be calculated (one pair at a time andthen summed) for positions 28, 31, and 50 in the antibody sequenceprovided in Table 2.

Methods for Targeted Affinity Enhancement

The present disclosure provides methods for the change of an amino acidresidue at a position in an antibody variable domain with other aminoacid residues to identify an amino acid change which results in theantibody variable domain having enhanced binding affinity for itsbinding partner. Enhanced binding affinity refers to a modified variabledomain that binds to a binding partner (e.g., antigen) with asignificantly higher equilibrium constant of association (K_(A)) orlower equilibrium constant of dissociation (K_(D)) than the parentvariable domain when the amounts of modified and parent variable domainsin the binding assay are the same. For example, the modified variabledomain with improved binding affinity may display at least 10%, at least15%, at least 25%, at least 50%, at least 75%, at least 100% (ortwo-fold), at least 5-fold, at least 8-fold, at least 10-fold, at least50-fold, at least 100-fold, or more, higher affinity to a bindingpartner than the corresponding parent variable domain. As used herein,binding partner refers to an antigen (e.g., an epitope on an antigen)recognized by an antibody or a molecular target of an antibody.

Amino acid residues in an antibody variable domain that are likely tocontribute to an antibody's binding affinity to a binding partner may bechanged to other amino acid residues to determine which change resultsin an enhancement of binding affinity. These residues may be changedwith other amino acid residues to generate a library or array ofmodified variable domains which may be selected for enhanced bindingaffinity to a binding partner as compared to the unmodified (parent)variable domain. These residues preferably include: alanine, arginine,asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine and valine. Cysteine and methionine may be includedbut are not preferred. In some embodiments cysteine and methionine arespecifically excluded.

In some embodiments, methods for targeted affinity enhancement mayutilize an amino acid residue's proximity group and/or conspicuousnessas described above, to select for those amino acid positions where anamino acid change is likely to enhance binding affinity.

An exemplary method for targeted affinity enhancement includes aligningmonoclonal antibody sequences with consensus or individual light-chainand heavy-chain sequences according to a standard numbering system suchas Kabat; optionally co-aligning with the antibody's own direct germlineprecursor sequences if they are known; optionally characterizing eachantibody position based upon the degree to which the residue differsfrom the corresponding consensus or germline residue in terms of chargeor size or chemical functionality; preferably changing one or morecontacting (C), supporting (S), peripheral (P) and/or interfacial (I)amino acid residues with other amino acids residues to produce a libraryor array of modified variable domains; and selecting those modifiedvariable domains that have enhanced affinity to a binding partnercompared to the unmodified variable domain. Less preferably one or moredistant (D) amino acid residues may additionally or alternatively bechanged.

Methods of Making Antibody Variable Domains With Enhanced BindingAffinity

Methods are provided for enhancing the binding affinity of an antibodyby means of producing targeted amino acid changes in the antibody'svariable domain. For example, engineered amino acid changes areintroduced at positions likely to produce enhanced affinity based uponan amino acid residue's proximity group.

In an exemplary method, amino acid changes are engineered at one or moreamino acid residues categorized as preferably contacting (C), peripheral(P), supporting (S) and/or interfacial on the “prox” lines of FIG. 2A,2B, 3A, 3B, 3C and/or 3D. In other embodiments, amino acid residuescategorized in more than one group may be selected for change. Lesspreferably one or more distant (D) amino acid residues may additionallyor alternatively be changed.

For example, methods are provided for making a modified variable domainof an antibody with enhanced binding affinity by modifying thenucleotide sequence of an antibody variable domain at a position thatpreferably encodes a contacting (C), peripheral (P), supporting (S)and/or interfacial (I) amino acid residue identified from the “prox”line as shown in FIG. 3A, 3B, 3C and/or 3D, thereby generating amodified antibody variable domain; and selecting a modified variabledomain that has enhanced binding affinity. Less preferably one or moredistant (D) amino acid residues may additionally or alternatively bechanged.

Methods are also provided for generating an array of modified antibodyvariable domains with eighteen amino acid changes at one or morecontacting (C) residues from a collection of modified variable domainsby obtaining a collection of modified antibody variable domainscontaining amino acid changes at one or more contacting (C) residues;sequencing the collection of modified variable domains; and arrangingeach sequenced modified antibody variable domain comprising one of theeighteen amino acid changes at one or more contacting (C) residue togenerate an array of modified variable domains with eighteen amino acidchanges at one or more contacting (C) residues.

Methods are provided for generating an array of modified variabledomains with eighteen amino acid changes at one or more contacting (C)residues by (a) synthesizing polynucleotides that encode sequences thatvary at one or more contacting (C) residues and contain eighteen aminoacid changes at each contacting (C) residue to generate modifiedantibody variable domains; and (b) arranging each synthesizedpolynucleotide from step (a) to generate an array of synthesizedpolynucleotides with eighteen amino acid changes at one or morecontacting (C) residues.

Methods are provided for generating an array of modified variabledomains with eighteen amino acid changes at one or more contacting (C)residues by (a) synthesizing polynucleotides that encode sequences thatvary at one or more contacting (C) residues and contain eighteen aminoacid changes at each contacting (C) residue to generate modifiedantibody variable domains; (b) transfecting each synthesizedpolynucleotide of step (a) separately into a host cell to generateclones comprising the synthesized polynucleotides; and (c) arrangingeach clone from step (b) to generate an array of clones capable ofexpressing modified variable domains with eighteen amino acid changes atone or more contacting (C) residues.

In some embodiments, one or more contacting residues to be changed maybe in complementarity determining domain-1 (CDR1) in a light chainvariable domain. In certain embodiments, the contacting residues may beat a position corresponding to position 28, 30 and/or 31 in CDR1.

In other embodiments, one or more contacting (C) residues to be changedmay be in CDR2 in a light chain variable domain. In certain embodiments,the contacting (C) residues may be at a position corresponding toposition 50, 51 and/or 53 in CDR2.

In some embodiments, one or more contacting (C) residues to be changedmay be in CDR1 in a heavy chain variable domain. In certain embodiments,the contacting (C) residues may be at a position corresponding toposition 32 and/or 33 in CDR1.

In some embodiments, one or more (C) contacting residues to be changedmay be in CDR2 in a heavy chain variable domain. In certain embodiments,the contacting (C) residues may be at a position corresponding toposition 50, 52, 53, 54, 56, and/or 58 in CDR2.

Modified variable domains are synthesized by modifying the nucleic acidof a parent variable domain, inserting the modified nucleic acid into anappropriate cloning vector and expressing the modified nucleic acid toproduce modified variable domains. Exemplary protocols are describedbelow.

1. Making Modified Variable Domain Nucleic Acids

Modified variable domains comprise one or more amino acid sequencechanges (e.g., substitutions) relative to a parent variable domainsequence to provide for enhanced binding affinity to a binding partnercompared to the parent variable domain.

In some embodiments, modified variable domains may have one or moreamino acid changes at preferably a contacting (C), peripheral (P),supporting (S) and/or interfacial (I) amino acid residue identified fromthe “prox” line as shown in FIG. 3A, 3B, 3C and/or 3D. Less preferablyone or more distant (D) amino acid residues may additionally oralternatively be changed. In some embodiments, a library of modifiedvariable domains may be constructed comprising 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 amino acid changes at preferablya contacting (C), peripheral (P), supporting (S), interfacial (I)residue and/or less preferably at a distant (D) amino acid residueidentified from the “prox” line as shown in FIG. 3A, 3B, 3C and/or 3D.

In some embodiments, an amino acid residue at preferably one or morecontacting (C), peripheral (P), supporting (S) and/or interfacial (I)amino acid residues, as identified from the “prox” line as shown in FIG.3A, 3B, 3C and/or 3D, may be changed with one or more of the followingpreferred amino acid residues: alanine, arginine, asparagine, asparticacid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosineand valine. Less preferably one or more distant (D) amino acid residuesmay additionally or alternatively be changed. In other embodiments, anamino acid residue at preferably a contacting (C), peripheral (P),supporting (S) and/or interfacial (I) amino acid residue, as identifiedfrom the “prox” line as shown in FIG. 3A, 3B, 3C and/or 3D is changedwith all of the following amino acid residues: alanine, arginine,asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine and valine. Less preferably one or more distant (D)amino acid residues may additionally or alternatively be changed.

In some embodiments, a modified variable domain may have two or moreamino acid changes at preferably a contacting (C), peripheral (P),supporting (S) and/or interfacial (I) amino residue identified from the“prox” line as shown in FIG. 3A, 3B, 3C and/or 3D. Less preferably oneor more distant (D) amino acid residues may additionally oralternatively be changed.

In some embodiments, a modified variable domain exhibits enhancedbinding affinity to a binding partner compared to the binding affinityexhibited by the parent variable domain. In some embodiments, a modifiedvariable domain exhibits at least a 10%, at least a 15%, at least a 25%,at least a 50%, at least a 75%, at least a 100% (or a two-fold), atleast a 5-fold, at least an 8-fold, at least a 10-fold, at least a50-fold, at least a 100-fold, or more, higher affinity to a bindingpartner than the corresponding parent variable domain.

A library and/or an array of modified variable domains may be generatedwhich contain multiple amino acid changes at a position of interest(e.g., at an amino acid residue in an antibody's variable domain atpreferably a contacting (C), peripheral (P), supporting (S) and/orinterfacial (I) amino acid residue as designated on the “prox” line ofFIG. 2A, 2B, 2C, 2D, 3A, 3B, 3C and/or 3D). Less preferably one or moredistant (D) amino acid residues may additionally or alternatively bechanged.

For example, amino acids may be preferably incorporated into a positionof interest by utilizing from three to nine different degenerate codons.Each degenerate codon will produce a mixture of from two to twelvedifferent oligonucleotides. One example of a seven degenerate primer setproduces basic amino acid changes\with a single primer that contains thedegenerate codon of ARG (R=A/G), encoding Arg/Lys. Polar amino acidchanges can be produced with two primers. For example, the first primercontains the degenerate codon WMC (W=A/T; M=A/C), encodingSer/Thr/Asn/Tyr, while the second polar primer utilizes the degeneratecodon CAS (S=C/G), encoding His/Gln. Acidic amino acid changes can beproduced with a single degenerate codon of GAS, encoding Glu/Asp.Non-polar functional amino acid changes can be produced with threeprimers: NTC (N=A/G/C/T), encoding Leu/Phe/Ile/Val, KGG (K=G/T),encoding Trp/Gly, and SCG, encoding Pro/Ala

An alternate substitution method may employ the use of three primerseach comprising a different degenerate codon to produce eighteen aminoacid changes. For example, the codons may include: NHT (where N=A/G/C/T,H=A/C/T), which codes forPhe/Ser/Tyr/Leu/Pro/His/Ile/Thr/Asn/Val/Ala/Asp; VAA (where V=A/C/G),which codes for Gln/Lys/Glu; and BGG (where B=C,G,T), which codes forTrp/Arg/Gly.

An alternate substitution method also may employ a nine degenerateprimer set by producing basic amino acid changes \with a single primerthat contains the degenerate codon of ARG (R=A/G), encoding Arg/Lys.Polar amino acid changes can be produced with three primers. Forexample, the first primer contains the degenerate codon WAC (W=A/T;M=A/C), encoding Asn/Tyr, while the second polar primer utilizes thedegenerate codon WCC, encoding Ser/Thr, while the third polar primerutilizes CAS (S=C/G), encoding His/Gln. Acidic amino acid changes can beproduced with a single degenerate codon of GAS, encoding Glu/Asp.Non-polar functional amino acid changes can be produced with five primersets: MTC (M=A/C), encoding Leu/Ile, KTC (K=G/T) encoding PheNal, KGG(K=G/T), encoding Trp/Gly, and SCG, encoding Pro/Alaln some embodiments,all seven of the degenerate primers are used to perform one PCRreaction. In other embodiments, each degenerate primer is used in aseparate PCR reaction. Any combination of PCR primers may be used in aPCR reaction.

DNA encoding modified variable domains may be prepared by a variety ofmethods known in the art. These methods include, but are not limited to,preparation by primer-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared modifiedvariable domain or parent variable domain. These techniques may utilizeantibody nucleic acid (DNA or RNA), or nucleic acid complementary to theantibody nucleic acid.

DNA encoding a modified variable domain with more than one amino acid tobe changed may be generated in one of several ways. If the amino acidsare located close together in the polypeptide chain, they may be mutatedsimultaneously using one primer that codes for all of the desired aminoacid changes. If, however, the amino acids are located some distancefrom each other (separated by more than about ten amino adds), it ismore difficult to generate a single primer that encodes all of thedesired changes. Instead, one of two alternative methods may beemployed.

In the first method, a separate primer is generated for each amino acidto be changed. The primers are then annealed to the single-strandedtemplate DNA simultaneously, and the second strand of DNA that issynthesized from the template will encode all of the desired amino acidchanges.

The alternative method involves two or more rounds of mutagenesis toproduce the desired mutant antibody. The first round is as described forthe modified variable domain which comprise one amino acid change:wild-type DNA is used for the template, a primer encoding the firstdesired amino acid change(s) is annealed to this template, and theheteroduplex DNA molecule is then generated. The second round ofmutagenesis utilizes the mutated DNA produced in the first round ofmutagenesis as the template. Thus, this template already contains one ormore mutations. The primer encoding the additional desired amino acidchange(s) is then annealed to this template, and the resulting strand ofDNA now encodes mutations from both the first and second rounds ofmutagenesis. This resultant DNA can be used as a template in a thirdround of mutagenesis, and so on.

2. Insertion of DNA into a Cloning Vehicle

The cDNA or genomic DNA encoding the modified antibody variable domainmay be inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. Many vectors areavailable, and selection of the appropriate vector will depend on 1)whether it is to be used for DNA amplification or for DNA expression, 2)the size of the DNA to be inserted into the vector, and 3) the host cellto be transformed with the vector. Each vector contains variouscomponents depending on its function (amplification of DNA or expressionof DNA) and the host cell for which it is compatible. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

For example, the cDNA or genomic DNA encoding the modified variabledomain may be inserted into a modified phage vector (i.e. phagemid).Construction of phage display libraries exploits the bacteriophage'sability to display peptides and proteins on their surfaces, i.e., ontheir capsids. Often, filamentous phage such as M13, f1 or fd are used.Filamentous phage contain single-stranded DNA surrounded by multiplecopies of genes encoding major and minor coat proteins, e.g., pIII. Coatproteins are displayed on the capsid's outer surface. DNA sequencesinserted in-frame with capsid protein genes are co-transcribed togenerate fusion proteins or protein fragments displayed on the phagesurface. Peptide phage libraries thus can display peptidesrepresentative of the diversity of the inserted genomic sequences.Significantly, these epitopes can be displayed in “natural” foldedconformations. The peptides expressed on phage display libraries canthen bind target molecules, i.e., they can specifically interact withbinding partner molecules such as antibodies (Petersen (1995) Mol. Gen.Genet. 249:425-31), cell surface receptors (Kay (1993) Gene 128:59-65),and extracellular and intracellular proteins (Gram (1993) J. Immunol.Methods 161:169-76).

The concept of using filamentous phages, such as M13, fd or fl, fordisplaying peptides on phage capsid surfaces was first introduced bySmith (1985) Science 228:1315-1317. Peptides have been displayed onphage surfaces to identify many potential ligands (see, e.g., Cwirla(1990) Proc. Natl. Acad. Sci. USA 87:6378-6382). There are numeroussystems and methods for generating phage display libraries described inthe scientific and patent literature (see, e.g., Sambrook and Russell,Molecule Cloning: A Laboratory Manual, 3rd edition, Cold Spring HarborLaboratory Press, Chapter 18, 2001; “Phage Display of Peptides andProteins: A Laboratory Manual, Academic Press, San Diego, 1996; Crameri(1994) Eur. J. Biochem. 226:53-58; de Kruif (1995) Proc. Natl. Acad.Sci. USA 92:3938-42; McGregor (1996) Mol. Biotechnol. 6:155-162;Jacobsson (1996) Biotechniques 20:1070-1076; Jespers (1996) Gene173:179-181; Jacobsson (1997) Microbiol Res. 152:121-128; Fack (1997) J.Immunol. Methods 206:43-52; Rossenu (1997) J. Protein Chem. 16:499-503;Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45; Rader (1997)Curr. Opin. Biotechnol. 8:503-508; Griffiths (1998) Curr. Opin.Biotechnol. 9:102-108).

Typically, exogenous nucleic acid to be displayed are inserted into acoat protein gene, e.g. gene III or gene VIII of the phage. Theresultant fusion proteins are displayed on the surface of the capsid.Protein VIII is present in approximately 2700 copies per phage, comparedto 3 to 5 copies for protein III (Jacobsson (1996), supra). Multivalentexpression vectors, such as phagemids, can be used for manipulation ofexogenous genomic or antibody encoding inserts and production of phageparticles in bacteria (see, e.g., Felici (1991) J. Mol. Biol.222:301-310).

Phagemid vectors are often employed for constructing the phage library.These vectors include the origin of DNA replication from the genome of asingle-stranded filamentous bacteriophage, e.g., M13, f1 or fd. Aphagemid can be used in the same way as an orthodox plasmid vector, butcan also be used to produce filamentous bacteriophage particle thatcontain single-stranded copies of cloned segments of DNA.

Other phage can also be used. For example, T7 vectors can be employed inwhich the displayed product on the mature phage particle is released bycell lysis.

In addition to phage epitope display libraries, analogous epitopedisplay libraries can also be used. For example, the methods of thedisclosure can also use yeast surface displayed epitope libraries (see,e.g., Boder (1997) Nat. Biotechnol. 15:553-557), which can beconstructed using such vectors as the pYD1 yeast expression vector.Other potential display systems include mammalian display vectors and E.coli libraries.

An antibody or antibody fragment, e.g., a scFv, Fab or Fv may bedisplayed on the surface of a phage using phage display techniques.Exemplary antibody phage display methods are known to those skilled inthe art and are described, e.g., in Hoogenboom, Overview of AntibodyPhage-Display Technology and Its Applications, from Methods in MolecularBiology: Antibody Phage Display: Methods and Protocols (2002) 178:1-37(O'Brien and Aitken, eds., Human Press, Totowa, N.J.). For example, alibrary or array of antibodies or antibody fragments (e.g., scFvs, Fabs,Fvs with an engineered intermolecular disulfide bond to stabilize theV_(H)-V_(L) pair, and diabodies) can be displayed on the surface of afilamentous phage, such as the nonlytic filamentous phage fd or M13.Antibodies or antibody fragments with the desired binding specificitycan then be selected.

An antibody phage-display library can be prepared using methods known tothose skilled in the art (see, e.g., Hoogenboom, Overview of AntibodyPhage-Display Technology and Its Applications, from Methods in MolecularBiology: Antibody Phage Display: Methods and Protocols (2002) 178:1-37(O'Brien and Aitken, eds., Human Press, Totowa, N.J.).

In some embodiments, cDNA is cloned into a phage display vector, such asa phagemid vector (e.g., pCES1, p XOMA Fab or pXOMA Fab-gIII). Incertain embodiments, cDNA encoding both heavy and light chains may bepresent on the same vector. In some embodiments, cDNA encoding scFvs arecloned in frame with all or a portion of gene III, which encodes theminor phage coat protein pIII. The phagemid directs the expression ofthe scFv-pIII fusion on the phage surface. In other embodiments, cDNAencoding heavy chain (or light chain) may be cloned in frame with all ora portion of gene III, and cDNA encoding light chain (or heavy chain) iscloned downstream of a signal sequence in the same vector. The signalsequence directs expression of the light chain (or heavy chain) into theperiplasm of the host cell, where the heavy and light chains assembleinto Fab fragments. Alternatively, in certain embodiments, cDNA encodingheavy chain and cDNA encoding light chain may be present on separatevectors. In certain embodiments, heavy chain and light chain cDNA may becloned separately, one into a phagemid and the other into a phagevector, which both contain signals for in vivo recombination in the hostcell.

The techniques for constructing and analyzing phage display librariesuses recombinant technology well known to those of skill in the art.General techniques, e.g., manipulation of nucleic encoding libraries,epitopes, antibodies, and vectors of interest, generating libraries,subcloning into expression vectors, labeling probes, sequencing DNA, DNAhybridization are described in the scientific and patent literature, seee.g., Sambrook and Russell, eds., Molecular Cloning: a Laboratory Manual(3rd), Vols. 1-3, Cold Spring Harbor Laboratory Press, (2001); CurrentProtocols in Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997-2001) (”Ausubel“); and, Laboratory Techniques inBiochemistry and Molecular Biology: Hybridization with Nucleic AcidProbes, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed.Elsevier, N.Y. (1993). Sequencing methods typically use dideoxysequencing, however, other methodologies are available and well known tothose of skill in the art.

3. Transformation of Host Cells

Suitable host cells for cloning or expressing the vectors herein mayinclude prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include eubacteria, such as Gram-negative or Gram-positiveorganisms, for example, E. coli, Bacilli such as B. subtilis,Pseudomonas species such as P. aeruginosa, Salmonella typhimurium, orSerratia marcescens.

For example, recombinant phagemid or phage vectors may be introducedinto a suitable bacterial host, such as E. coli. In some embodimentsusing phagemid, the host may be infected with helper phage to supplyphage structural proteins, thereby allowing expression of phageparticles carrying the antibody-pIII fusion protein on the phagesurface.

Methods for Identifying an Antibody Variable Domain Having EnhancedAffinity for a Binding Partner

Methods are provided for identifying a modified antibody variable domainhaving enhanced binding affinity for a binding partner by contacting aparent antibody variable domain with the binding partner underconditions that permit binding; contacting modified antibody variabledomains made by the methods of the present disclosure with the bindingpartner under conditions that permit binding; and determining bindingaffinity of the modified antibody variable domains and the parentantibody variable domain for the binding partner, wherein modifiedantibody variable domains that have a binding affinity for the bindingpartner greater than the binding affinity of the parent antibodyvariable domain for the binding partner are identified as havingenhanced binding affinity.

Isolated antibody variable domains may exhibit binding affinity assingle chains, in the absence of assembly into a heteromeric structurewith their respective V_(H) or V_(L) subunits. As such, populations ofV_(H) and V_(L) altered antibody variable domains can be expressed aloneand screened for binding affinity having substantially the same orgreater binding affinity compared to the parent antibody V_(H) or V_(L)variable domain.

Alternatively, populations of antibody V_(H) and V_(L) altered variabledomains polypeptides can be co-expressed so that they self-assemble intoheteromeric altered antibody variable domain binding fragments. Theheteromeric binding fragment population can then be screened for speciesexhibiting enhanced binding affinity to a binding partner compared tothe binding affinity of the parent antibody variable domain.

The expressed population of modified antibody variable domains can bescreened for the identification of one or more altered antibody variabledomain species which exhibit enhanced binding affinity to a bindingpartner as compared with the parent antibody variable domain. Screeningcan be accomplished using various methods well known in the art fordetermining the binding affinity of a polypeptide or compound.Additionally, methods based on determining the relative affinity ofbinding molecules to their partner by comparing the amount of bindingbetween the modified antibody variable domain and the binding partnercan similarly be used for the identification of species exhibitingbinding affinity substantially the same or greater than the parentantibody variable domain to the binding partner. The above methods canbe performed, for example, in solution or in solid phase. Moreover,various formats of binding assays are well known in the art and include,for example, immobilization to filters such as nylon or nitrocellulose;two-dimensional arrays, enzyme linked immunosorbant assay (ELISA),radioimmuno-assay (RIA), panning and plasmon resonance (see, e.g.,Sambrook et al., supra, and Ansubel et al., supra).

For the screening of populations of polypeptides such as the modifiedantibody variable domains produced by the methods of the disclosure,immobilization of the modified antibody variable domains to filters orother solid substrates is particularly advantageous because largenumbers of different species can be efficiently screened for binding toa binding partner. Such filter lifts allow for the identification ofmodified antibody variable domains that exhibit enhanced bindingaffinity compared to the parent antibody variable domain to the bindingpartner. Alternatively, the modified antibody variable domains may beexpressed on the surface of a cell or bacteriophage. For example,panning on an immobilized binding partner can be used to efficientlyscreen for the relative binding affinity of species within thepopulation of modified antibody variable domains and for those whichexhibit enhanced binding affinity to the binding partner than the parentantibody variable domain.

Another affinity method for screening populations of modified antibodyvariable domains is a capture lift assay that is useful for identifyinga binding molecule having selective affinity for a ligand. This methodemploys the selective immobilization of modified antibody variabledomains to a solid support and then screening of the selectivelyimmobilized modified antibody variable domains for selective bindinginteractions against the binding partner. Selective immobilizationfunctions to increase the sensitivity of the binding interaction beingmeasured since initial immobilization of a population of modifiedantibody variable domains onto a solid support reduces non-specificbinding interactions with irrelevant molecules or contaminants which canbe present in the reaction.

Another method for screening populations or for measuring the affinityof individual modified antibody variable domains is through surfaceplasmon resonance (SPR). This method is based on the phenomenon whichoccurs when surface plasmon waves are excited at a metal/liquidinterface. Light is directed at, and reflected from, the side of thesurface not in contact with sample, and SPR causes a reduction in thereflected light intensity at a specific combination of angle andwavelength. Biomolecular binding events cause changes in the refractiveindex at the surface layer, which are detected as changes in the SPRsignal. The binding event can be either binding association ordisassociation between a receptor-ligand pair. The changes in refractiveindex can be measured essentially instantaneously and therefore allowsfor determination of the individual components of an affinity constant.More specifically, the method enables accurate measurements ofassociation rates (k_(on)) and disassociation rates (k_(off)).

Measurements of k_(on) and k_(off) values can be advantageous becausethey can identify modified antibody variable domains with enhancedbinding affinity for a binding partner. For example, a modified antibodyvariable domain can be more efficacious because it has, for example, ahigher k_(on) valued compared to the parent antibody variable domain.Increased efficacy is conferred because molecules with higher k_(on)values can specifically bind and inhibit their binding partner at afaster rate. Similarly, a modified antibody variable domain can be moreefficacious because it exhibits a lower k_(off) value compared tomolecules having similar binding affinity. Increased efficacy observedwith molecules having lower k_(off) rates can be observed because, oncebound, the molecules are slower to dissociate from their bindingpartner.

Methods for measuring the affinity, including association anddisassociation rates using surface plasmon resonance are well known inthe arts and can be found described in, for example, Jonsson andMalmquist, Advances in Biosensors, 2:291-336 (1992) and Wu et al. Proc.Natl. Acad. Sci. USA, 95:6037-6042 (1998).

Using any of the above described screening methods, a modified antibodyvariable domain having binding affinity substantially the same orgreater than the parent variable domain is identified by detecting thebinding of at least one altered variable domain within the population toits binding partner.

Detection methods for identification of species within the population ofmodified variable domains can be direct or indirect and can include, forexample, the measurement of light emission, radioisotopes, calorimetricdyes and fluorochromes. Direct detection includes methods that operatewithout intermediates or secondary measuring procedures to assess theamount of the binding partner bound by the modified antibody variabledomain. Such methods generally employ ligands that are themselveslabeled by, for example, radioactive, light emitting or fluorescentmoieties. In contrast, indirect detection includes methods that operatethrough an intermediate or secondary measuring procedure. These methodsgenerally employ molecules that specifically react with the bindingpartner and can themselves be directly labeled or detected by asecondary reagent. For example, a modified antibody variable domainspecific for a binding partner can be detected using an antibody capableof interacting with the modified antibody variable domain, again usingthe detection methods described above for direct detection. Indirectmethods can additionally employ detection by enzymatic labels. Moreover,for the specific example of screening for catalytic antibodies, thedisappearance of a substrate or the appearance of a product can be usedas an indirect measure of binding affinity or catalytic activity.

In some embodiments, the modified antibody variable domain has a bindingaffinity for the binding partner greater than the binding affinity ofthe parent variable domain for the binding partner and thus isidentified as having enhanced binding affinity.

In some embodiments, a modified antibody variable domain exhibitsenhanced binding affinity to a binding partner compared to the bindingaffinity between the parent variable domain and the binding partner. Insome embodiments, a modified variable domain exhibits an at least 10%,at least 15%, at least 25%, at least 50%, at least 75%, at least 100%(or two-fold), at least 5-fold, at least 8-fold, at least 10-fold, atleast 50-fold, at least 100-fold, or more, higher affinity to a bindingpartner than the corresponding parent antibody variable domain.

In other embodiments, the modified antibody variable domain has abinding affinity for the binding partner less than the binding affinityof the parent antibody variable domain for the binding partner and thusis identified as having reduced binding affinity for the bindingpartner.

This disclosure is further illustrated by the following examples whichare provided to facilitate the practice of the disclosed methods. Theseexamples are not intended to limit the scope of the disclosure in anyway.

Examples Example 1 Design of Primers for Synthesis of Nucleic AcidEncoding Modified Antibody Variable Domains

Each contacting residue identified from the “prox” lines in FIG. 2, 3A,3B, 3C and/or 3D may be changed with other amino acid residues (e.g.,alanine, arginine, asparagine, aspartic acid, glutamine, glutamine acid,glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline,serine, threonine, tryptophan, tyrosine and valine) by performing PCRwith an oligonucleotide containing one of seven different degeneratecodons (e.g., ARG (where R=A/G), WMC (where W=A/T and M=A/C), CAS (whereS=C/G), GAS (where S=C/G), NTC (where N=A/G/C/T), KGG (where K=G/T) andSCG (where S=C/G)).

In an exemplary substitution method, use of seven primers, eachcomprising one of the seven degenerate codons, may be employed to changeone or more contacting (C) amino acid positions in a parent nucleic acidmolecule to 18 other amino acid residues. An alternate substitutionmethod may employ the use of three primers each comprising a differentdegenerate codon to produce eighteen amino acid changes at one or morecontacting resides in a parent nucleic acid molecule. For example, thecodons may include: NHT (where N=A/G/C/T, H=A/C/T), which codes forPhe/Ser/Tyr/Leu/Pro/His/Ile/Thr/Asn/Val/Ala/Asp; VAA (where V=A/C/G),which codes for Gln/Lys/Glu; and BGG (where B=C,G,T), which codes forTrp/Arg/Gly. This allows production of all eighteen amino acids at equalratios if oligonucleotides comprising NHT is used at a 4:1:1 ratio witholigonucleotides comprising VAA and oligonucleotides comprising BGG,since NHT encodes twelve amino acids and VAA and BGG both encode threeamino acids.

Primers containing one or more degenerate codons may be used tointroduce a desired class of amino acid residue at a contacting (C)position by hybridizing to a parent nucleic acid (e.g., the nucleotidesequence encoding the degenerate codon pairs with a contacting (C)position to be changed). Basic amino acid changes can be produced at acontacting (C) position with a single oligonucleotide that contains thecodon mixture of ARG (R=A/G), encoding Arg/Lys. Further, polar aminoacid changes can be introduced at a contacting (C) position with twooligonucleotides. The first oligonucleotide contains the codon mixtureWMC (W=A/T; M=A/C), encoding Ser/Thr/Asn/Tyr, while the secondoligonucleotide utilizes the codon mixture CAS (S=C/G), encodingHis/Gln. Additionally, acidic amino acid changes may be introduced at acontacting (C) position with a single codon mixture of GAS, encodingGlu/Asp. Last, non-polar amino acid changes may be introduced at acontacting (C) position with a mixture of three primers with degeneratecodons: NTC (N=A/G/C/T), encoding Leu/Phe/Ile/Val, KGG (K=G/T), encodingTrp/Gly, and SCG, encoding Pro/Ala.

Example 2 Construction of a Library Containing Modified AntibodyVariable Domains

Modified antibody variable domains containing amino acid changes at oneor more contacting (C) residues present within an exemplary antibody,for example, ING-1 (a mouse-human chimeric antibody containing the Br-1mouse variable region domains and human constant regions domains whichselectively binds to Ep-CAM (U.S. Pat. No. 5,576,184), heavy chainsequence represented by SEQ ID NO: 579, light chain sequence representedby SEQ ID NO: 580) may be synthesized by PCR amplification from a parentnucleic acid molecule using synthetic oligonucleotides containing adegenerate codon (SEQ ID NO: 1-285 or SEQ ID NO: 583-699). Similarly,modified antibody variable domains containing amino acid changes at oneor more contacting (C) residues present within an exemplary antibody,for example, IL-1 antibody (heavy chain sequence represented by SEQ IDNO: 581, kappa chain sequence represented by SEQ ID NO: 582) may besynthesized by PCR amplification from a parent nucleic acid moleculeusing synthetic oligonucleotides containing a degenerate codon (SEQ IDNO: 286-578 or SEQ ID NO: 700-806).

For example, each library primer containing the degenerate codondescribed above for ING-1 may be used in a PCR reaction to synthesize aDNA fragment which incorporates an amino acid change and a 3′restriction site. In an exemplary method, PCR may be conducted at acontacting (C) position (e.g., H3-3) by utilizing the CDRH3oligonucleotide H3-3NP2 (SEQ ID NO: 267):5′-GCTACATATTTCTGTGCAAGATTTGGCTCTKGGGTGGACTACTGGGGTCAAG G-3′, and thereverse primer Notl-R (SEQ ID NO: 285):5′-AGCGGCCGCACAAGATTTGGGCTCAACTCTC-3′) (see, FIG. 5) under standardconditions (see, e.g., Sambrook and Russell, Molecule Cloning: ALaboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press,2001). After PCR amplification, fragments are obtained which compriseeither a tryptophan or glycine residue at the internal codon (underlinedabove). Further, six other PCR reactions may be performed at the H3-3position, utilizing SEQ ID NO: 285 with one of SEQ ID NOs: 262-266 and268 under the conditions described above to obtain other amino acidchanges at the site. Next, the products from these reactions may becombined at equal mass, except for reactions which used SEQ ID NO: 263and 266 as a primer (this mixture is termed the pooled H3-3 library).Due to the degeneracy of these primers, twice the mass of the sampleobtained with SEQ ID NO: 263 and 266 is added to produce an equimolarratio of encoded amino acids.

An additional PCR reaction may be performed to create a fragment (calledthe H3-R fragment) which contains a 5′ restriction site and anoverlapping complementary region to the library fragments describedabove. As an example, for the H3-3 position, a PCR reaction may beperformed utilizing the Asc-F2 (SEQ ID NO: 284) and one of the H3R (SEQID NO: 247) primer. The 3′ portion of this molecule contains a regionthat is identical to the 5′ portion of the molecules created above whichpermits the use of a PCR reaction to create a contiguous moleculecontaining a 5′ and 3′ restriction site.

A PCR reaction may be performed to fuse the above PCR products togetherinto a single molecule. Products from the two PCR reactions describedabove may be melted and re-annealed to allow for the region of overlapfrom the two molecules to hybridize. For example, an equal mass of thepooled H3-3 library (approximately two uL of each pooled PCR reaction)and the H3-R fragment may be annealed at their regions of overlap. Next,amplification of annealed molecules with both the Asc-F2 primer (SEQ IDNO: 284) and the Notl-R primer (SEQ ID NO: 247) allows for the synthesisof a single contiguous molecule (see, e.g., Sambrook and Russell,Molecule Cloning: A Laboratory Manual, 3rd edition, Cold Spring HarborLaboratory Press, 2001).

The DNA fragment synthesized by the methods above may be cloned into apXOMA Fab or pXOMA Fab-gIII vector. Briefly, the DNA fragment ispurified by using the QIAGEN® PCR purification kit and sequentiallydigesting the fragment with Notl (NEW ENGLAND BIOLABS®, Ipswich, Mass.)and Ascl (NEW ENGLAND BIOLABS®, Ipswich, Mass.) (See, Methods inMolecular Biology, vol. 178: Antibody Phage Display: Methods andProtocols Edited by: P. M. O'Brien and R. Aitken, Humana Press,“Standard Protocols for the Construction of Fab Libraries, Clark, M. A.,39-58) (see, e.g., FIG. 6). Next, the vectors may be ligated with themutagenized insert using T4 Ligase (NEW ENGLAND BIOLABS®, Ipswich,Mass.) and transformed into TG1 cells by electroporation.

Example 3 Selection of High Affinity Binders

Phage containing a modified antibody variable domain that binds to anantigen (e.g., Ep-Cam or IL-1β) with high affinity may be selected bystandard panning protocols (see, e.g., Methods in Molecular Biology,vol. 178: Antibody Phage Display: Methods and Protocols Edited by: P. M.O'Brien and R. Aitken, Humana Press, “Panning of Antibody Phage-DisplayLibraries”, Coomber, D. W. J. pp 133-145, and “Selection of AntibodiesAgainst Biotinylated Antigens”, Chames, P. et al. p. 147-157).

In an exemplary method, library phage for the panning procedure areamplified by inoculating fifty milliliters of 2YT with library TG1 cellsand grown to an OD₆₀₀ of 0.6-0.8. Helper phage VCSM13 are added to theinoculated 2YT culture at a multiplicity of infection (M.O.I.) of 10(e.g., in 50 mL of cells with OD₆₀₀=0.6 there are 0.6×3⁸×50=9×10⁹ cells,M.O.I. of 10 is therefore 9¹⁰ helper phage, which corresponds to about10 μl of 1¹³ stock phage). The helper phage are used to infect the TG1cells by gently mixing the phage with the cells with no shaking forthirty minutes. The culture is then shaken for an additional thirtyminutes at 180 rpm. Following infection, the culture is spun down at2500 rpm for ten minutes. The resulting cell pellet is resuspended infifty milliliters of 2TYAmpKan and grown overnight at 30° C. and thesupernatant is removed and discarded.

Exemplary methods of panning include coating one well of a NUNC®MAXISORP plate with fifty μl of Ep-Cam or IL-1β at 0.1 μg/ml inDULBECCO'S® PBS with Calcium and Magnesium chloride (Invitrogen,Carlsbad, Calif.) and incubating the plates overnight at 4° C. The wellsare then blocked with 5% milk in PBS for one hour at room temperature.Separately 0.5 ml of phage supernatant from the overnight culturedescribed above are blocked with 300 μL of 10% milk in PBS for one hourat room temperature. Blocked phage (e.g., approximately 200 μl) areadded to the blocked wells in 3% BSA-PBS and incubated at roomtemperature with shaking for one to two hours. After incubation, thewells are emptied and washed five times with PBST quick wash (e.g.,PBS+0.05% Tween 20), then washed five times with PBST five minute wash,followed by five washes with PBS quick wash and lastly washed five timeswith PBS five minute wash. Phage bound to the wells are eluted byincubating with 200 μL/well of freshly prepared 100 mM TEA (prepared byadding 140 μL of 7.18 M Triethylamine stock to ten ml H₂O for 20 minutesat room temperature (see, e.g., Sambrook and Russell, Molecular Cloning:A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press,2001). The eluate is transferred to a Falcon tube containing 0.5 ml 2MTRIS-HCl pH 7.4. The pH of the eluate is checked with pH paper to ensurethat it is about pH 7 and adjusted if necessary.

Eluted phage from the exemplary panning method are amplified byinfecting TG1 cells. In an exemplary method, TG1 cells are grown to anOD₆₀₀=0.6 (e.g., mid log phase) and ten ml of the culture is added tothe phage eluate from the panning method described above. The elutedphage are used to infect the TG1 cells at 37° C. for thirty minuteswithout shaking and then continued for an additional thirty minutes at37° C. with shaking at 240 rpm. After the infection, the culture iscentrifuged at 2500 rpm for five minutes. Next, the supernatant isremoved and the cell pellet is resuspended in 700 μL of 2YTAG. There-suspension is plated on two 15 cm 2YTAG agar plates and incubated at30° C. overnight. After the overnight incubation, the cells are scrapedfrom the two plates using five to ten milliliters of 2YTAG per plate,and transferred to a fifty milliliter falcon tube where they are used tomake a glycerol stock.

In an alternative exemplary method, panning may be performed withbiotinylated Ep-Cam or IL-1β. Briefly, two hundred microliters ofstreptavidin beads (Dynal) are blocked in 5% BSA-PBS (100 μl of theblocked beads are used for the de-selection and 100 μL for theselection). Using a magnet, the beads are removed from the 5% BSA-PBSand rinsed twice in PBS. To the rinsed beads is added one milliliter of5% BSA-PBS and the beads are incubated at room temperature for one hourwith very gentle rotation. After the incubation, the beads are splitinto two tubes, with the supernatant removed from one tube for thede-selection. Phage solution is added to the tube with beads designatedfor the de-selection and resuspended. The phage-bead solution isincubated at room temperature for forty-five minutes with gentlerotation. After the incubation, the phage supernatant (de-selected phagesolution) is transferred to a new tube using a magnet. Next, thede-selected phage solution is incubated at room temperature for sixtyminutes with one hundred pmols of biotinylated Ep-Cam or IL-1β. Thephage-biotinylated Ep-Cam or IL-1β solution is then added to a newaliquot of streptavidin beads (with the supernatant removed) andincubated at room temperature for sixty minutes. After the incubation,the beads are separated from the supernatant using a magnet. Next thebeads are washed five times with one ml of 0.5% BSA-PBST by adding thewash to the tube, closing the tube and resuspending the pellet, puttingback in the magnet waiting a few seconds until the beads are attached tothe magnet side of the tube and removing the wash with a pipetman.Further, the beads are washed five times in 0.5% BSA-PBST for fiveminutes for each wash, washed five times with one milliliter of 0.5%BSA-PBS, washed five times for five minutes each wash in fivemilliliters of 0.5% BSA, and washed one time with PBS. Bound phage areeluted by incubating the beads with 500 μL of freshly prepared 100 mMTEA (add 140 μL of 7.18 M Triethylamine stock to 10 ml H₂O) for thirtyminutes at room temperature with gentle rotation. The eluate isseparated from the beads by using a magnet and transferred to a fiftymilliliter falcon tube containing 250 μl of 1M TRIS pH 7.4 to neutralizethe TEA and can be used for infection and/or amplification (see, e.g.,Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rdedition, Cold Spring Harbor Laboratory Press, 2001). For example, logphase TG1 cells may be infected with phage eluate at 37° C. for one hourat ninety rpm. After infection of the cells, the culture is centrifugedat 2500 rpm for five minutes and the supernatant removed. Next, the cellpellet is resuspended in 700 μl of 2YTAG, plated onto two 15 cm2YT-ampicillin-2% glucose agar plates and incubated at 30° C. overnight.

Example 4 Screening of an Affinity Matured Antibody Using the DELFIA®Competition Assay

Individual Fabs obtained from the affinity-based selection of librariesof the ING-1 antibody clone are tested for their ability to inhibit thebinding of Ep-Cam to the parent chimeric ING-1 IgG antibody using acompetitive screening assay (e.g., the microplate based competitivescreening DELFIA® assay (PERKIN ELMER®, Waltham, Mass.)). Ninety-sixwell plates containing two hundred and fifty microliters of 2YT mediaare inoculated with glycerol stock of Fab-expressing E. coli transformedwith the pXOMA-Fab vector. The culture is grown at 37° C. until cloudy(approximate OD600=0.5) and inoculated with IPTG to a finalconcentration of 1 mM. The cultures are grown overnight at 30° C. Inaddition, a Costar plate 3922 (White) is coated with 1.25 ug/mL ofparental ING-1 chimeric IgG O/N at 4° C.

Periplasmic extracts (PPE) of the overnight expression constructs areprepared by spinning the overnight expression plates at 3000 rpm forfifteen minutes, discarding supernatant and adding 60 microliters of PPBbuffer (periplasmic extraction buffer, 30 mM Tris-HCl pH 8.0, 20%sucrose, 1 mM EDTA) to each well. The pellets are resuspended, and 90microliters of cold PPB diluted 1:5 with cold water are added to eachwell. This mixture is incubated on ice for one hour and subsequentlyspun down at 3000 rpm for fifteen minutes. This PPE supernatant istransferred to a new plate. The PPE is diluted into 10% PPE in PBS, 5%PPE in PBS, and 1% PPE in PBS. For the coated Costar plate, it is washedthree times with PBS-tween and blocked with 350 microliters of 3% BSA inPBS for one hour.

The blocked Costar plate is washed three times with PBS and thenbiotinylated Ep-Cam is added to the diluted PPE to a final concentrationof 3 nM. The diluted PPE and biotinylated Ep-Cam solution is then addedto the coated Costar plate and incubated for one and a half hours atroom temperature. The plates are washed three times with PBST and fiftymicroliters of 1:250 dilution of Europrium-Streptavidin in Delfia AssayBuffer (PERKIN ELMER®, Waltham, Mass.) is added. The mixture isincubated at room temperature for one hour, and the Time-ResolvedFluorescence Plate reader is setup (Gemini microplate reader, MolecularDevices), interval 200-1600 microseconds, 20 reads/well, excitation 345nm, emission 618 nm and cutoff 590 nm. The plates are washed seven timeswith Delfia Wash Buffer (PERKIN ELMER®, Waltham, Mass.), followed by theaddition of fifty μl of Delfia Enhancement buffer (PERKIN ELMER®,Waltham, Mass.) and incubated for five minutes. The plates are read onthe Gemini plate reader. Plates with decreased signal compared withcontrol parental antibody show greater binding by the affinity maturedFab and can be further characterized by Biacore (e.g., Biacore 2000 orA100) and other affinity measuring techniques (see, e.g., Tables 4 and5).

Similarly, XPA23 antibody clones may be tested for their ability toinhibit the binding of IL-1β to the parent chimeric XPA23 IgG using acompetitive screening assay as described above.

TABLE 4 Delfia Screening of 10% Periplasmic Extract 1 2 3 4 5 6 7 8 9 1011 12 A 46.9 37.1 71.2 75.7 51.3 22.3 65.8 72.9 58.8 81.7 56.2 96.7 B2.6 55.2 39.2 54.8 31.7 41.3 57.1 56.7 21.6 77.8 1.8 102.0 C 53.2 42.372.5 61.2 16.2 78.0 41.2 57.2 63.8 28.6 13.6 100.7 D 49.0 45.5 8.9 1.021.5 82.8 105.8 67.3 68.5 61.8 63.5 100.6 E 49.1 72.1 68.6 0.3 91.8 57.653.1 8.3 58.3 60.4 82.2 −0.4 F 61.7 72.1 71.8 45.6 44.6 53.1 15.3 73.284.7 15.1 59.0 0.1 G 58.4 26.4 1.0 59.4 62.3 19.9 −0.1 49.0 52.4 76.246.8 0.3 H 36.1 67.7 65.2 27.4 34.3 50.3 60.0 60.1 56.8 83.0 49.3 −0.4Percentage of inhibition is shown in each well using the average signalfrom wells A12-D12 as positive control, 100% inhibition and the averagesignal in well E12-H12 as 0% inhibition negative control wells. Wellsbolded show strong competition in the Delphia assay.

TABLE 5

Percentage of inhibition is shown in each well using the average signalfrom wells A12-D12 as positive control, 100% inhibition and the averagesignal in well E12-H12 as 0% inhibition negative control wells. Wellsbolded show strong competition in the Delphia assay. Boxed wells retainstrong inhibition and are prioritized for affinity testing.

Example 5 Screening of an Affinity Matured Antibody Using KineticTitration Analysis

Kinetic properties of affinity matured antibodies, for example, asrepresented by XPA23 clones such as Y208L may be determined by kinetictitration analysis. In an exemplary method, an antigen such as IL-1β isamine coupled to a CM5 sensor chip. Each sample (e.g., from lowest tohighest concentration) may be injected for 240 seconds at a flow rate of30 μl/min at a selected temperature (e.g., 25° C.). Sample are allowedto dissociate for 30 seconds except the highest concentration which maybe permitted 300 seconds to dissociate. The assay is run at 25° C.

Biaevaluation software (e.g., Biacore 2000 evaluation software) is usedto calculate dissociation rates of individual samples and the relativeamount of sample bound to each test surface. The data is fit to anappropriate kinetic model (e.g., the kinetic titration model). Forexample, XPA23 had a ka=2.5e5 and a kd=1.2e−2 KD=4.6e−8, while themodified XPA23 Y208L mutant had a ka=3.57E+05 kd=5.80E−03 KD=1.62E−08.

Example 6 ELISA Measurement for Fab Expression or Antigen Binding

Additionally or alternatively to the Biacore assay described below inExample 10, an ELISA assay may be used for the identification ofmodified antibody variable domains that bind its binding partner or forverifying expression of Fab domains.

In an exemplary method, ELISA plates (e.g., Nunc MAXISORP™) are coatedwith 1 μg/ml EpCam, 1 μg/mL EpCam for EpCam ELISA, 1 μg/mL IL-1(Peprotech), or anti-human IgG, F(ab′)₂ fragment specific antibody(Jackson Immunoresearch) in PBS at 50 μg/ml. The ELISA plates are thencovered and incubated at 4° C. overnight. After the incubation, thecoated ELISA plates are washed three times with PBS. The plates are thenfilled with 370 μl of 3% milk (e.g., Carnation, nonfat) and incubatedfor one hour at room temperature. Separately, 150 μl of periplasmicextract is blocked by adding 50 μl of 15% milk and incubating theextract for one hour at room temperature. The blocked plates are washedthree times with PBS and 50 μL of the blocked periplasmic extract isadded to each well of the antigen coated ELISA plates. The plates areincubated for two hours at room temperature and then washed four timeswith TBST.

Secondary antibodies are added to each ELISA plate. For the Ep-Cam orIL-1 ELISA, 50 μl of mouse anti-human c-myc antibody (9E10 Ab, Roche) at2.5 μg/ml in 3% milk is added to each well. For the anti-Fab ELISA, 50μl of biotin-SP-conjugated anti-human IgG F(ab′)2 fragment specificantibody (Jackson lmmunoresearch) at 1:2000 dilution in 3% milk is addedto each well. The plates from both ELISAs are incubated at roomtemperature for one hour. After the incubation, the plates are washedfour times with TBST. After the washes, a tertiary antibody may be addedto the plates in both ELISAs. For the Ep-Cam or IL-1 ELISA, 50 μl ofgoat anti-mouse IgG-HRP (Pierce) diluted 1:10,000 in 3% milk is added toeach well. For the anti-Fab ELISA, 50 μl of extravidin-HRP conjugate(Sigma) at a 1:500 dilution in 3% milk is added to each well. Again theplates from both ELISAs are incubated for one hour at room temperature.After the incubation, the plates are washed four times with TBST. Next,50 μl of the TMB substrate (Calbiochem) is added to each well andincubated until the color develops (do not incubate long enough to seethe negative control turn blue). The reaction is stopped by adding 50 μlof 2N H₂SO₄ to each well and the plates are read at 450 nm.

Example 7 Methods for Off-Rate Ranking of Antibodies or FragmentsThereof

A high-throughput off-rate ranking method is used for rapidprioritization of modified antibody variable domains that bind to theirbinding partner by analyzing their relative off-rates (using, e.g.,Biacore 2000 or A100).

In an exemplary method, modified antibody variable domains (e.g.,Epcam-binding) are produced in ninety-six well plates by inoculating twohundred and fifty microliters of 2YT media with a glycerol stock ofFab-expressing E. coli transformed with a pXOMA-Fab vector comprising amodified Epcam-binding variable domain. The culture is grown at 37° C.until cloudy (e.g., approximate OD₆₀₀=0.5), inoculated with IPTG to afinal concentration of 1 mM and grown overnight at 30° C.

Next, periplasmic extracts (PPE) of the overnight expression constructsare prepared by spinning the overnight expression plates at 3000 rpm forfifteen minutes, discarding the supernatant and adding 60 μl of PPBbuffer to each well. The pellets are resuspended, and 90 μl of cold PPBdiluted 1:5 with cold water is added to each well. This mixture isincubated on ice for one hour and subsequently spun down at 3000 rpm forfifteen minutes. The supernatant is transferred to a new plate and theperiplasmic extracts are used for the Biacore (e.g., Biacore 2000 orA100) determination.

Epcam from the periplasmic extracts is amine coupled (e.g., 10 μpg/mLEpcam in pH 4.5 acetate, seven minute injection at 5 μl/minute) to a CM5sensor chip and periplasmic extracts containing the antibody fragmentsare injected over the sensor, resulting in binding of the Fab to theimmobilized Epcam. Non specific binding of the antibody fragment to thesensor surface is corrected by subtracting the interaction of theantibody fragment with a blank flow cell (e.g., having no immobilizedEpcam) from the interaction of the antibody fragment with the Epcamimmobilized flow cell. The instrument settings are: a flow rate of 20microliters/minute, an injection time of three minutes, a dissociationtime of five minutes and an instrument temperature set to 25° C.Biaevaluation software is used to calculate dissociation rates ofindividual samples and the relative amount of sample bound to each testsurface. Samples are then ranked according to their dissociation rates.Sensograms depicting the off-rates for heavy chains (FIG. 15) and lightchains (FIG. 16) are shown. The off rates for the improved clones aretabulated for the heavy chain (FIG. 11) and the light chain (FIG. 12).

Likewise, modified XPA23 variable domains (e.g., IL-1β-binding) may beranked according to their dissociation rates using the high-throughputoff-rate ranking method described above. The instrument settings are: aflow rate of 30 microliters/minute, an injection time of three minutes,a dissociation time of ten minutes and an instrument temperature set to25° C. The off rates for the improved clones are tabulated for the heavychain (FIG. 13) and the light chain (FIG. 14).

The modified antibody variable domains of the present disclosure mayhave a k_(off) that is greater than (see, e.g., FIG. 20), less than(see, e.g., FIG. 18) or equal to (see, e.g., FIG. 19) than an unmodifiedantibody variable domain.

Example 8 Reformatting of Candidate Clones to IgG

Two of the improved off-rate clones from the k_(off) analysis werereformatted into IgG₁ format by PCR amplification of the heavy and lightchain variable domains and cloning the PCR amplified regions into amammalian expression vector containing the Fc and the light chainconstant domain respectively. The heavy chain is cloned into a mammalianexpression vector containing a CMV promoter using Bsml and Nhel sitesfor the 5′ and 3′ ends respectively and is cloned in frame with theheavy chain secretion signal on the 5′ end and the constant CH1,CH2, andCH3 portions of the IgG molecule on the 3′ end. The amplificationsequences are as follows: (ING-HC-IgGF5′-ATATATTGCATTCCCAGATCCAGTTGGTGCAGTC-3′), ING-HC-IgGR(5′-ATATATGCTAGCTGAGCTGACGGTGACCGAGGTTCC-3′). The light chain is clonedinto a similarly constructed expression vector utilizing a blunt 5′cloning site and the BsiWI site on the 3′ end and is cloned in framewith the light chain secretion signal on the 5′ end and the light chainconstant region on the 3′ end. The PCR amplification primer sequencesare as follows: (ING-LC-IgGF 5′-CAAATTGTGATGACGCAGGC-3′) and(ING-LC-IgGR 5′-ATATATCGTACGTTTCATCTCTAGTTTGGTGCC-3′). The PCRs areperformed under standard conditions: see, e.g., Sambrook and Russell,Molecule Cloning: A Laboratory Manual, 3rd edition, Cold Spring HarborLaboratory Press, 2001. Improved off-rate clones reformatted into IgG,vectors are transiently co-transfected in a 2:1 light chain to heavychain DNA ratio into HEK 292 cells using Lipofectamine 2000 (Invitrogen)using the manufacturer's guidelines. Secreted IgGs secreted from HEK 292cells are purified using protein A SEPHAROSE® (GE-AMERSHAM® Piscataway,N.J.) using the manufacturer's guidelines and tested by BIACORE® (e.g.,Biacore 2000 or A100) for affinity (see, e.g., FIGS. 11 and 15) andExample 8.

Example 9 Expression and Testing of Modified Antibody Variable Domainswith a Combination of Amino Acid Changes

Modified antibody variable domains with improved off-rates andaffinities as compared to a parent variable region may be identified byemploying the DELFIA® competition assay and/or BIACORE® (e.g., Biacore2000 or A100) off-rate ranking. Clones with improved k_(off) aresequenced and aligned by both their light and heavy chain. Identifiedamino acid changes in the light and heavy chain that increase affinitycan be combined in one modified antibody variable domain for potentialadditive and synergistic combinations. Modifications for combination mayutilize the residues that have improved off-rates greater than or equalto 4.9 fold compared with the parental antibodies (see, e.g., FIGS. 11,12). For any given amino acid position, the change that leads to thegreatest improvement is chosen for study. This compilation is describedin Table 6, and will lead to 21 combinations of heavy and light chains(e.g., 7 heavy chains combined in all variations with three lightchains).

TABLE 6 Heavy and Light Chain CDR1, CDR2 and/or CDR3 Combinations CDR1CDR2 CDR3 Heavy Chain Combinations G33F wt wt wt T53I wt wt wt G100RG33F T53I wt wt T53I G100R G33F wt G100R G33F T53I G100R Light ChainCombinations wt Q55R wt wt wt E98T wt Q55R E98T

Alternatively, the initial modifications for combination may utilize theresidues that have improved off-rates greater than or equal toapproximately 2.5-fold compared with the parental antibodies (see, e.g.,FIGS. 13, 14). For any given amino acid change, the change that leads tothe greatest improvement is chosen for study. The amino acids withgreater than or equal to approximately 2.5 fold improved k_(off) arecompiled in Table 7. There are two amino acids in CDR1 (position 28),two amino acids in position 100, three amino acids in position 101, andfive amino acids in position 102. In all, there are 60 (2×2×3×5=60)combinations.

TABLE 7 Heavy Chain CDR1 and CDR3 Combinations CDR1 CDR3 28T (wt) 100G(wt) 28I 100R 101S (wt) 101I 101G 102A (wt) 102Y 102F 102W 102G

A PCR based strategy may be used to create a modified antibody lightchain containing more than one amino acid change (see, e.g., FIG. 7). Inan exemplary method, PCR may be used to amplify three segments of theV_(k) gene, two of which may be engineered to contain an amino acidchange. For example, to create a light chain containing the mutationsQ55R and E98T, PCR product 1 may be synthesized using the HindIII-F (SEQID NO: 814) and L2R primer (SEQ ID NO: 74), PCR product 2 may besynthesized using L2-Q55R primer (SEQ ID NO: 808) and the L3R primer(SEQ ID NO: 110) and PCR product 3 may be synthesized using L3-E98Tprimer (SEQ ID NO: 807)and the Ascl-R primer (SEQ ID NO: 812). The PCRproducts are then melted and re-annealed such that their regions ofoverlap hybridize. Subsequently, all three PCR products may be joinedinto one molecule by PCR amplification using the forward primer from PCRproduct 1 (HindIII-For) (SEQ ID NO: 814) and the reverse primer from PCRproduct 3 (Ascl-R) (SEQ ID NO: 812). In an exemplary method to create aheavy chain containing the mutations outlined above and described inFIG. 7, product 1 may be synthesized using the Ascl-F (SEQ ID NO: 813)and H1R primer (SEQ ID NO: 146), PCR product 2 may be synthesized usingH1-28TI primer and the H3R primer (SEQ ID NO: 247) and PCR product 3 maybe synthesized using each H3 combination primer (6 primers, 6 r×ns) andthe Notl-R primer (SEQ ID NO: 285). The PCR products are then melted andre-annealed such that their regions of overlap hybridize. Subsequently,all three PCR products may be joined into one molecule by PCRamplification using the forward primer from PCR product 1 (Ascl-F) (SEQID NO: 813) and the reverse primer from PCR product 3 (Notl-R) (SEQ IDNO: 285).

In an exemplary method, a 50 μL PCR reaction for the production of PCRproduct 1, 2 and 3 may be performed with 25 pmol of each of the forwardand reverse primers, 10 ng of template DNA, 5 μL PFU buffer, 2.5 μL of10 μM dNTPs, 1 μL PFU and water to 50 μL. The PCR reaction is heated to94° C. for two minutes, followed by 25 cycles of 30 seconds at 94° C.,30 seconds at 54° C., and one minute at 72° C. After the 25 cycles, afinal 72° C. incubation may be performed for five minutes.

An equal mass of the three PCR products may be combined in a PCRreaction to produce a modified variable domain with several amino acidchanges which enhance affinity. Briefly, the PCR may be conducted byadding approximately 2 μL of each pooled PCR reaction to 5 μL PFUbuffer, 25 pmol of both HindIII-f primer (SEQ ID NO: 814)and Ascl-Rprimers (SEQ ID NO: 812), 2.5 μL of 10 μM dNTPs, 1 μL PFU polymerase andwater to 100 μL. Next, the PCR reaction is heated to 94° C. for twominutes, followed by twenty-five cycles of thirty seconds at 94° C., 30seconds at 54° C., and finally one minute at 72° C. After the twentycycles, a final 72° C. incubation is performed for five minutes.

The resulting DNA fragment may be purified (e.g., using the QIAGEN® PCRpurification kit (Valencia, Calif.)) and sequentially digested withHindIII (NEB) and then Ascl (NEW ENGLAND BIOLABS®, Ipswich, Mass.) suchthat it may be cloned into the pXOMA Fab or pXOMA Fab-gIII vector.

For the heavy chain modifications, a similar PCR based strategy may beused to create a modified antibody heavy chain containing more than oneamino acid change (see, e.g., FIG. 8). In an exemplary method, PCR maybe used to amplify four segments of the V_(H) gene, three of which maybe engineered to contain the G33F, T53I and G100R amino acid changes.For example, PCR product 1 may be synthesized using the Ascl-F (SEQ IDNO: 813) and H1R primers (SEQ ID NO: 146), PCR product 2 may besynthesized using the H1-G33F primer (SEQ ID NO: 809) and H2R primer(SEQ ID NO: 182), PCR product 3 may be synthesized using H2-T3I primer(SEQ ID NO: 810) and H3R primer (SEQ ID NO: 247) and PCR product 4 maybe synthesized using H3-G100R primer (SEQ ID NO: 811) and the Notl-Rprimer (SEQ ID NO: 285). The PCR products are then melted andre-annealed such that their regions of overlap hybridize. All four PCRproducts may then be joined into one molecule by PCR amplification usingthe forward primer from PCR product 1 (Ascl-F) (SEQ ID NO: 813) and thereverse primer from PCR product 3 (Notl-R) (SEQ ID NO: 285).

In an exemplary method, a 50 μL PCR reaction for the production of PCRproducts 1, 2, 3 and 4 may be performed with 25 pmol each of the forwardand reverse primers, 10 ng of template DNA, 5 μL PFU buffer, 2.5 μL of10 μM dNTPs, 1 μL PFU and water to 50 μL. The PCR reaction is heated to94° C. for 2 minutes, followed by 25 cycles of 30 sec at 94° C., 30seconds at 54° C., and one minute at 72° C. After the 25 cycles, a final72° C. incubation may be performed for five minutes.

An equal mass of the four PCR products may be combined in a PCR reactionto produce a modified variable domain with several amino acid changeswhich enhance affinity. Briefly, the PCR may be conducted by addingapproximately 2 μL of each pooled PCR reaction to 5 μL PFU buffer, 25pmol of both Ascl-F primer (SEQ ID NO: 813) and Notl-R primer (SEQ IDNO: 285), 2.5 μL of 10 μM dNTPs, 1 μL PFU polymerase and water to 100μL. Next, the PCR reaction is heated to 94° C. for two minutes, followedby twenty-five cycles of thirty seconds at 94° C., 30 seconds at 54° C.,and finally one minute at 72° C. After the twenty cycles, a final 72° C.incubation is performed for five minutes.

The heavy chain PCR fragments and the vector will be digested with Ascl(NEW ENGLAND BIOLABS®, Ipswich, Mass.) and Notl (NEW ENGLAND BIOLABS®,Ipswich, Mass.) such that it may be cloned into the pXOMA Fab or pXOMAFab-gIII vector.

Example 10 Biacore Measurement of IgG Affinity

IgGs that bind Epcam in Example 7 are tested by BIACORE® for affinity(see, e.g., FIG. 15). For example, kinetic analysis of anti-Epcam mAb'sare conducted on a Biacore 2000®.

In an exemplary method, the ING1 antibody is diluted to 0.5 μg/mL inHBS-EP running buffer and injected for two minutes at 5 μl/ minute overa high density protein A/G surface. Next, six serial 3 fold dilutions ofEpcam are prepared in running buffer and injected in triplicate inrandom order over the high density protein A/G surface with bufferinjections evenly distributed throughout the run. The sample injectionsare then double referenced against the blank flow cells and bufferinjections to correct for any bulk shift or non-specific binding. Dataare then analyzed with the Biaevaluation software from Biacore andsensorgrams are fit utilizing the 1:1 langmuir model (see, e.g., FIG.15).

Example 11 Construction of Arrays of Modified Antibody Variable Domains

Arrays of modified antibody variable domains (e.g., modified ING-1variable domains) with amino acids changes at desired positions (e.g.,contacting (C) residues) may be generated and tested for enhancedbinding affinity compared to the parent variable domain (e.g., ING-1).Modified variable domains used in the array may be obtained directlyfrom a library of modified variable domains as described in Example 2 ormay first be screened for those modified variable domains that exhibitenhanced binding as compared to the parent variable domain as describedin Examples 3, 4 and 5.

In an exemplary method, each contacting (C) residue in the heavy andlight chain variable region of ING-1 is separately changed (e.g., by PCRmutagenesis) with alanine, arginine, asparagine, aspartic acid,glutamine, glutamine acid, glycine, histidine, isoleucine, leucine,lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosineand valine to generate modified ING-1 variable domains. CDNAs encodingthe modified ING-1 variable domains are then inserted into a pXOMAvector and used to transform electrocompetent TG1 cells. The clones areplated on 2YT-Amp₁₀₀/2% Glucose plates (Teknova) and the plates filledwith 250 μl of 2YT-Amp₁₀₀/well (Teknova). Each well is inoculated with asingle colony comprising a single amino acid change at a contacting (C)residue. The colonies are grown by incubating the plates at 37° C. fortwo to four hours with shaking at 450 rpm. After the incubation, theplates are duplicated to sequencing plates by filling new deep-wellculture plates (Thomson) with one milliliter of 2YT-Amp₁₀₀Gluc₂%/wellfrom the grown cultures. The Genetix 96-pin replicator is used totransfer cells from the master plate to the new sequencing plates. Thesequencing plates are grown overnight at 37° C. with shaking at 450 rpm.After the incubation, the sequencing plate is spun down at 5000 rpm forten minutes and the supernatant is discarded. Samples from the plate aresequenced (e.g., samples may be submitted for automated miniprep andautomated sequencing (Elim biopharmaceuticals). After the incubation,Master Plates are made by adding glycerol to a final concentration of15% to the wells on the glycerol plate and storing the plates at −80° C.The unique clones and their well position in the master plate areidentified after sequencing results are returned.

Eighteen different clones, each containing an amino acid change at acontacting (C) residue in ING-1, are identified (typically 96 sequencedclones yield all eighteen clones). Unique clones from the master platesare rearrayed to a new 96-well master plate containing 2YT-Amp₁₀₀ bytransferring ten microliters of glycerol stock from the master plate tothe rearrayed master plate. Alternatively, automation, such as the QPIXII is used to transfer the glycerol stock containing the unique clonesto the new master plate. The new rearrayed glycerol master plates arereplicated into new expression plates to perform Biacore (e.g., BiacoreA100) analysis (see, e.g., Table 8 and Table 9). Arrays may also beconstructed for XPA 23 modified antibodies (see, e.g., Table 10 and 11).

TABLE 8 Biacore Analysis of Modified Light Chain Variable Regions^(1, 2 3) NP Aromatic Neg Pos Polar D E R K H Y W F Q N CDR1 K 27 1.26−1.00 1.26 ? 1.06 nd −1.00 1.62 1.52 −1.00 S 28 1.63 1.02 2.78 2.32 1.902.02 nd 2.38 1.99 −1.00 L 29 −1.00 −1.00 −1.00 nd nd −1.00 −1.00 −1.00−1.00 1.85 L 30 1.47 −1.00 −1.00 −1.00 1.45 1.53 −1.00 1.56 1.60 1.45 H31 0.71 0.68 0.06 0.05 0.95 2.16 1.66 nd 0.57 0.50 S 32 0.94 nd 1.791.32 1.13 1.37 1.27 1.64 1.10 −1.00 N 33 0.49 0.65 0.71 0.70 0.73 0.801.04 0.93 0.73 1.38 I 35 0.19 0.16 0.92 0.61 0.59 0.51 0.34 0.66 0.410.50 T 36 0.05 1.60 −1.00 1.15 0.79 nd 1.30 nd 1.04 0.74 Y 37 nd 0.01 nd0.02 4.07 0.95 0.85 0.63 0.02 0.06 CDR2 Y 54 0.03 0.05 −1.00 3.62 −1.000.92 0.96 0.94 1.23 −1.00 Q 55 0.05 0.05 5.31 0.46 3.82 nd 4.11 0.860.95 0.36 M 56 1.36 0.71 0.92 0.98 1.32 1.21 1.29 1.40 1.12 0.99 S 570.95 0.93 1.17 1.54 1.01 −1.00 2.34 0.96 −1.00 1.17 N 58 nd 0.97 1.771.40 1.16 1.43 1.99 1.03 1.55 0.95 CDR3 L 97 −1.00 0.75 −1.00 0.61 0.420.98 1.59 0.93 0.91 0.48 E 98 1.62 0.98 3.08 2.22 1.23 1.23 1.10 1.431.41 −1.00 L 99 0.02 0.01 0.04 0.02 0.05 1.00 0.89 0.43 0.02 2.00 P 1000.02 0.06 0.05 0.03 0.05 1.94 1.51 1.65 0.04 0.05 R 101 −1.00 −1.00 0.930.04 −1.00 −1.00 −1.00 −1.00 −1.00 −1.00 NP Polar Aliphatic Small NP S TV I L A C G P M CDR1 K 27 −1.00 −1.00 nd 1.15 1.39 1.10 nd 1.11 1.29 ndS 28 1.08 1.19 2.15 2.35 2.60 1.53 nd nd 2.42 nd L 29 2.03 nd nd −1.00 ?1.97 nd 1.53 −1.00 nd L 30 1.43 1.89 1.69 0.96 0.97 1.27 nd 1.64 0.82 ndH 31 0.82 1.94 1.17 1.14 0.59 1.26 nd 0.73 1.19 nd S 32 ? nd 0.93 1.171.12 1.35 nd 1.39 0.83 nd N 33 0.48 0.60 −1.00  nd nd 0.76 nd 0.72 0.67nd I 35 0.55 0.87 1.08 nd 0.60 0.46 nd 0.39 0.70 nd T 36 1.10 0.94 0.980.76 1.64 1.02 nd 1.09 0.67 nd Y 37 0.09 −1.00 −1.00  0.90 nd 0.04 nd1.30 nd nd CDR2 Y 54 0.90 0.61 1.32 −1.00 3.44 0.08 nd 1.85 0.86 nd Q 550.56 0.66 1.53 1.42 0.71 0.64 nd 0.70 0.95 nd M 56 1.05 0.80 −1.00  1.370.74 0.86 nd 0.80 1.38 nd S 57 1.00 0.86 0.89 0.98 1.38 1.15 nd −1.00 ndnd N 58 1.65 1.42 2.84 2.51 1.47 1.79 nd 1.87 3.47 nd CDR3 L 97 0.790.54 1.44 2.62 0.93 0.50 nd −1.00 0.95 nd E 98 −1.00 4.90 nd nd 2.821.35 nd 1.63 −1.00 nd L 99 0.04 0.09 1.04 2.07 0.93 1.43 nd 0.02 0.01 ndP 100 0.06 0.08 0.14 0.14 0.03 0.05 nd 1.62 1.01 nd R 101 −1.00 −1.00−1.00  −1.00 1.33 −1.00 nd −1.00 −1.00 nd ¹ A value of −1 indicates nobinding ² Bolded values indicate the highest affinity o affinity (asmeasured by how many “fold” differences in affinity. The mutant is incomparison to original, e.g., 2.0 as twice as strong and 0.5 as half asstrong) obtained for an amino acid change at the position ³ nd indicatesthat binding affinity was not determined

TABLE 9 Biacore Analysis of Modified Heavy Chain Variable Regions^(1, 2, 3) NP Aromatic Neg Pos Polar D E R K H Y W F Q N CDR1 T 28 0.981.23 1.23 1.77 1.15 1.86 1.08 1.28 −1.00 0.69 T 30 0.63 0.73 1.39 0.94nd 2.00 1.26 nd nd 0.73 K 31 0.66 0.54 0.76 0.96 0.78 1.00 1.02 nd nd−1.00 Y 32 nd 0.08 0.43 0.08 0.60 0.84 nd 1.05 0.10 nd G 33 −1.00 −1.000.03 −1.00 0.02 6.16 −1.00 7.19 0.06 −1.00 CDR2 W 50 3.27 −1 0.10 0.040.02 0.04 0.97 0.09 0.01 0.03 N 52 0.02 −1 −1 −1 0.02 −1 −1 −1 −1 0.98 T53 −1 −1 −1 1.79 −1 −1 −1 −1 −1 −1  Y 54 0.05 0.07 3.72 3.62 1.00 0.920.96 0.65 0.66 2.11 T 55 0.03 −1 0.14 0.45 0.05 0.03 0.10 0.03 0.03 0.17E 56 0.81 0.95 1.34 1.27 1.74 1.04 1.17 0.78 1.23 1.01 E 57 1.17 1.071.71 nd 1.16 1.37 1.39 1.06 −1.00 1.57 P 58 0.54 0.44 nd 1.14 nd 0.991.11 0.98 1.11 0.90 T 59 0.87 0.51 1.22 1.43 0.40 nd 2.24 0.43 −1.000.96 CDR3 G 100 −1 −1 7.51 1.59 −1 −1 −1 −1 −1.00 1.55 S 101 0.21 0.76nd 2.20 1.35 1.79 1.22 1.16 2.18 0.97 A 102 0.28 0.51 2.18 1.48 2.403.01 3.13 2.97 1.01 0.94 D 104 nd 0.14 −1 −1 −1 −1 −1 −1 −1 0.73 Y 105−1 −1 0.66 −1 0.94 nd 0.84 0.91 0.87 nd NP Polar Aliphatic Small NP S TV I L A C G P M CDR1 T 28 1.07 nd 2.08 2.45 nd 1.56 nd 0.92 2.16 nd T 300.91 0.93 1.30 nd 1.26 0.89 nd −1.00 0.93 nd K 31 0.49 nd 1.41 1.17 0.600.39 nd 1.02 nd nd Y 32 0.01 nd 0.11 0.03 0.05 0.01 nd 0.02 0.01 nd G 33nd 0.01 0.04 0.55 2.27 0.06 nd nd 6.31 nd CDR2 W 50 0.02 0.03 0.02 0.070.04 0.03 nd 0.03 −1 nd N 52 −1 −1 −1 −1    −1 −1 nd −1 −1 nd T 53 0.171.19 2.44 11.40  nd 9.03 nd −1 −1 nd Y 54 0.49 0.36 1.32 0.28 1.80 0.47nd 3.72 0.86 nd T 55 0.42 nd 0.28 nd 0.95 nd nd 0.02 nd nd E 56 1.461.21 0.86 0.85 0.64 1.67 nd 1.37 0.01 nd E 57 1.41 1.44 nd 1.34 −1 1.65nd 1.45 −1.00 nd P 58 1.07 1.00 1.01 nd 0.64 1.03 nd 1.31 1.06 nd T 59nd 0.99 1.03 0.76 0.95 nd nd 0.35 −1.00 nd CDR3 G 100 1.68 0.61 2.17 nd0.65 1.99 nd nd −1 nd S 101 nd 0.84 1.92 3.53 −1 1.15 nd 3.31 nd nd A102 0.94 0.68 1.20 0.79 −1 nd nd 3.58 0.87 nd D 104 1.87 −1 −1 nd −1 −1nd 0.41 −1 nd Y 105 0.09 0.12 0.18 0.23 0.22 0.08 nd −1.00 −1 nd ¹ Avalue of −1 indicates no binding ² Bolded values indicate the highestaffinity o affinity (as measured by how many “fold” differences inaffinity. The mutant is in comparison to original, e.g., 2.0 as twice asstrong and 0.5 as half as strong) obtained for an amino acid change atthe position ³ nd indicates that binding affinity was not determined

TABLE 10 Biacore Analysis of Modified C5A (XPA23) Light Chain VariableRegions ^(1, 2, 3) NP Aromatic Neg Pos Polar D E R K H Y W F Q N CDR1Q27 1.07 1.08 0.89 −1.00 1.09 nd −1.00 2.96 1.06 −1.00 D28 1.00 0.740.94 0.82 1.23 1.45 3.81 1.43 nd 1.00 N30 0.81 0.64 0.74 0.61 1.00 1.401.06 1.59 0.60 1.08 R31 11.11 −1.00 1.06 1.18 −1.00 −1.00 0.43 0.92 0.270.38 W32 −1.00 −1.00 −1.00 −1.00 −1.00 −1.00 0.99 −1.00 −1.00 −1.00 CDR2H49 0.11 −1.00 0.21 0.10 1.10 0.52 0.21 0.50 −1.00 0.31 S50 −1.00 0.02−1.00 0.10 0.05 0.05 0.02 −1.00 0.21 0.25 A51 0.13 0.29 0.18 nd 0.610.45 0.24 −1.00 0.24 0.30 T52 0.72 0.61 3.37 3.23 0.91 1.01 0.87 1.050.83 0.83 S53 −1.00 1.13 3.29 4.07 nd 1.23 1.11 1.24 1.09 1.23 CDR3 A911.08 0.10 −1.00 nd 1.12 −1.00 −1.00 −1.00 −1.00 0.10 D92 0.83 0.99 −1.000.23 0.63 −1.00 0.12 0.26 0.56 0.67 S93 4.59 3.71 0.91 0.86 1.08 1.455.49 1.32 1.54 3.76 F94 −1.00 −1.00 −1.00 −1.00 −1.00 −1.00 −1.00 1.14−1.00 −1.00 P95 1.32 0.88 −1.00 −1.00 −1.00 −1.00 −1.00 0.98 −1.00 −1.00L96 −1.00 −1.00 −1.00 −1.00 0.07 −1.00 4.49 0.28 0.21 0.06 NP PolarAliphatic Small NP S T V I L A C G P M CDR1 Q27 3.30 0.64 −1.00 0.820.83 1.47 nd −1.00 −1.00 nd D28 5.25 1.19 0.85 −1.00 9.64 0.88 nd 1.211.17 nd N30 0.72 0.97 0.89 0.65 0.68 0.61 nd 0.71 0.69 nd R31 0.44 0.240.44 0.68 0.51 8.48 nd 0.92 −1.00 nd W32 −1.00 −1.00 −1.00 −1.00 −1.00−1.00 nd −1.00 −1.00 nd CDR2 H49 0.14 −1.00 −1.00 −1.00 0.07 0.23 nd−1.00 0.86 nd S50 1.04 0.87 0.21 0.25 0.49 0.13 nd 0.14 −1.00 nd A510.95 0.76 0.31 −1.00 0.16 1.09 nd 3.84 0.13 nd T52 0.78 1.02 0.82 0.800.78 0.66 nd −1.00 1.08 nd S53 1.08 0.95 1.42 1.42 0.99 1.07 nd 1.034.92 nd CDR3 A91 0.90 0.67 0.51 −1.00 0.39 −1.00 nd −1.00 −1.00 nd D926.59 0.34 −1.00 0.33 0.14 0.40 nd 0.26 −1.00 nd S93 1.20 1.47 3.81 1.351.08 1.16 nd 0.75 −1.00 nd F94 −1.00 −1.00 0.20 0.88 0.55 −1.00 nd −1.000.17 nd P95 4.05 −1.00 −1.00 −1.00 −1.00 3.83 nd −1.00 −1.00 nd L96 0.230.06 nd 0.67 1.16 0.26 nd −1.00 −1.00 nd ¹ A value of −1 indicates nobinding ² Bolded values indicate the highest affinity (as measured byhow many “fold” differences in affinity. The mutant is in comparison tooriginal, e.g., 2.0 as twice as strong and 0.5 as half as strong)obtained for an amino acid change at the position ³ nd indicates thatbinding affinity was not determined

TABLE 11 Biacore Analysis of Modified C5A (XPA23) Heavy Chain VariableRegions ^(1, 2, 3) NP Aromatic Neg Pos Polar D E R K H Y W F Q N CDR1T28 nd −1.00 −1.00 −1.00 −1.00 nd −1.00 nd −1.00 nd S30 −1.00 −1.00 0.10−1.00 −1.00 −1.00 0.77 −1.00 −1.00 0.15 K31 0.04 −1.00 0.90 1.30 1.110.04 0.84 0.05 −1.00 −1.00 Y32 0.68 0.12 nd nd 0.62 nd nd 0.75 −1.000.04 F33 0.92 0.86 −1.00 0.85 −1.00 0.77 0.79 −1.00 0.78 0.97 F35 0.06−1.00 −1.00 0.68 −1.00 0.85 −1.00 −1.00 −1.00 0.86 CDR2 V50 −1.00 −1.00−1.00 −1.00 −1.00 −1.00 −1.00 −1.00 −1.00 0.03 I51 0.07 0.73 0.10 0.110.08 1.75 −1.00 0.90 0.83 0.68 S52 −1.00 −1.00 −1.00 −1.00 −1.00 −1.00−1.00 −1.00 −1.00 −1.00 P53 −1.00 0.03 nd 0.04 −1.00 0.03 0.05 0.05 0.030.06 S54 0.12 0.03 1.43 1.53 nd 0.99 1.07 0.87 1.01 0.08 G55 −1.00 0.110.95 0.11 nd 0.08 nd nd 0.90 0.10 G56 0.05 1.21 2.11 2.05 0.82 1.27 1.511.41 1.31 1.06 M57 −1.00 −1.00 0.06 0.04 0.08 0.86 nd 1.03 0.02 0.03 T580.12 −1.00 1.14 −1.00 0.98 1.01 0.93 1.04 −1.00 0.90 R59 −1.00 −1.00−1.00 0.94 −1.00 −1.00 0.09 0.92 0.86 0.86 CDR3 V99 nd −1.00 nd −1.00 ndnd nd 0.03 −1.00 −1.00 G100 −1.00 nd −1.00 −1.00 0.06 0.09 0.05 −1.00−1.00 nd Y101 −1.00 0.03 0.85 nd 0.04 1.00 0.81 0.85 0.06 0.06 G102 nd−1.00 −1.00 −1.00 −1.00 −1.00 −1.00 nd −1.00 1.17 G103 −1.00 −1.00 −1.00−1.00 nd nd −1.00 −1.00 −1.00 nd N104 −1.00 −1.00 −1.00 −1.00 0.07 −1.00−1.00 −1.00 0.03 1.02 S105 nd 1.17 −1.00 nd 0.05 −1.00 −1.00 1.25 0.980.85 D106 0.98 0.04 0.06 0.04 0.02 −1.00 −1.00 −1.00 0.02 0.07 Y107 0.850.90 0.85 0.82 −1.00 −1.00 0.89 0.90 0.87 −1.00 NP Polar Aliphatic SmallNP S T V I L A C G P M CDR1 T28 nd −1.00 nd nd nd nd nd −1.00 nd nd S300.85 0.06 −1.00 0.08 0.91 0.77 nd 0.13 −1.00 nd K31 −1.00 −1.00 0.810.74 1.08 nd nd nd −1.00 nd Y32 1.33 0.80 1.07 nd 0.75 −1.00 nd 1.000.68 nd F33 0.73 0.78 0.76 0.75 0.88 0.75 nd 0.76 0.91 nd F35 0.09 0.740.89 0.85 0.78 0.07 nd 0.04 0.06 nd CDR2 V50 −1.00 0.03 0.19 0.10 0.090.09 nd 0.03 −1.00 nd I51 0.69 0.86 0.99 0.95 0.79 1.04 nd 0.94 −1.00 ndS52 nd 0.04 0.04 −1.00 −1.00 0.05 nd 0.03 0.05 nd P53 0.03 nd 1.10 0.080.04 0.05 nd 0.02 0.94 nd S54 nd 0.14 1.00 0.86 0.91 1.00 nd 1.43 0.06nd G55 1.02 nd nd 0.86 0.08 nd nd 0.99 nd nd G56 nd 1.11 nd 1.71 1.411.21 nd nd 0.85 nd M57 0.04 0.06 0.03 0.03 0.06 0.03 nd nd −1.00 nd T580.82 1.04 0.99 0.95 0.95 0.14 nd 0.95 1.81 nd R59 0.13 0.76 −1.00 −1.000.08 −1.00 nd 1.04 0.07 nd CDR3 V99 −1.00 0.10 nd nd −1.00 0.03 nd nd−1.00 nd G100 2.80 0.12 nd nd −1.00 nd nd 1.02 −1.00 nd Y101 nd 0.060.09 0.83 1.91 0.04 nd −1.00 0.04 nd G102 nd nd −1.00 nd −1.00 −1.00 ndnd −1.00 nd G103 nd nd 2.56 nd nd 0.03 nd 0.93 nd nd N104 1.02 0.95 1.27nd 1.19 1.61 nd 1.21 nd nd S105 1.09 0.11 0.08 0.07 −1.00 0.84 nd 0.121.41 nd D106 0.03 −1.00 −1.00 −1.00 −1.00 −1.00 nd 0.03 −1.00 nd Y1070.89 0.83 0.93 0.96 0.86 0.83 nd 0.10 0.04 nd ¹ A value of −1 indicatesno binding ² Bolded values indicate the highest affinity o affinity (asmeasured by how many “fold” differences in affinity. The mutant is incomparison to original, e.g., 2.0 as twice as strong and 0.5 as half asstrong) obtained for an amino acid change at the position ³ nd indicatesthat binding affinity was not determined

Example 12 Construction of Arrays of Modified Antibody Variable Domains

Arrays of modified antibody variable domains (e.g., modified ING-1variable domains) with amino acids changes at desired positions (e.g.,contacting (C) residues) may be generated and tested for enhancedbinding affinity compared to the parent variable domain (e.g., ING-1).Modified variable domains used in the array may be obtained directlyfrom a library of modified variable domains as described in Example 2 ormay first be screened for those modified variable domains that exhibitenhanced binding as compared to the parent variable domain as describedin Examples 3, 4 and 5.

In an exemplary method, each contacting (C) residue in the heavy andlight chain variable region of ING-1 is separately changed (e.g., by PCRmutagenesis) with alanine, arginine, asparagine, aspartic acid,glutamine, glutamine acid, glycine, histidine, isoleucine, leucine,lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosineand valine to generate modified ING-1 variable domains. cDNAs encodingthe modified ING-1 variable domains are then inserted into a pXOMAvector and used to transform electrocompetent TG1 cells. The clones areplated on 2YT-Amp₁₀₀/2% Glucose plates (Teknova) and the plates filledwith 250 μl of 2YT-Amp₁₀₀/well (Teknova). Each well is inoculated with asingle colony comprising a single amino acid change at a contacting (C)residue. The colonies are grown by incubating the plates at 37° C. fortwo to four hours with shaking at 450 rpm. After the incubation, theplates are duplicated to expression plates by filling new plates(Costar) with two hundred and fifty microliters of 2YT-Amp100 media(Teknova). The Genetix 96-pin replicator is used to transfer cells fromthe Master plate to the new expression plates. The culture is grown at37° C. until cloudy (e.g., approximate OD₆₀₀=0.5), inoculated with IPTGto a final concentration of 1 mM and grown overnight at 30° C.

Next, periplasmic extracts (PPE) of the overnight expression constructsare prepared by spinning the overnight expression plates at 3000 rpm forfifteen minutes, discarding the supernatant and adding 60 μl of PPBbuffer to each well. The pellets are resuspended, and 90 μl of cold PPBdiluted 1:5 with cold water is added to each well. This mixture isincubated on ice for one hour and subsequently spun down at 3000 rpm forfifteen minutes. The supernatant is transferred to a new plate and theperiplasmic extracts are used for the Biacore (e.g., Biacore A100)determination.

After Biacore determination, wells that contain clones with improved offrates are sequenced and further characterized (e.g. IgG reformatting andaffinity determination).

Example 13 Affinity Optimization of an Antibody Variable Domain byTargeted Mutagenesis of Selected Amino Acid Residues

Affinity optimized antibodies or fragments thereof may be obtained bymutation of one or more selected amino acid residues in a parentantibody or binding fragment thereof with other amino acid residues(e.g., alanine, arginine, asparagine, aspartic acid, glutamine, glutamicacid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine or valine). Methods foroptimization of an antibody variable domain may comprise the stages asset forth below.

A. Selection of Amino Acid Residues for Mutation

Amino acid residues at one or more positions in a parent antibody orbinding fragment thereof are selected for mutagenesis. Such methods mayinclude, for example, identifying the proximity assigned to amino acidpositions in the variable domain of the antibody using the “prox” lineas shown in FIGS. 3A, 3B, 3C and/or 3D. One or more amino acid residuesidentified as C, P, S and/or I residues may be selected for mutation.

B. Design of Primers for Mutagenesis

Primers are designed to mutagenize a parent nucleic acid sequence thatcodes for an antibody or binding fragment thereof.

For a PCR-based mutagenesis method, a primer may be designed such thatthe forward primer sequence flanks both sides (e.g., 20 base pairs) ofthe position to be mutated. Additionally, it is preferred that theprimer be 70 bases or less in length. A representative CDR comprisingamino acid residues 1-8 is shown below.

aa#                      1   2  3  4   5  6  7   8                         G   F  T  F   S  K  Y   F5′-G TCTTTCTTGC GCTGCTTCCG GATTCACTTT CTCTAAGTAC TTTATGTTTT(SEQ ID NO: 964) GGGTTCGCCAAGC-3′3′-C AGAAAGAACG CGACGAAGGC CTAAGTGAAA GAGATTCATG AAATACAAAA(SEQ ID NO: 965) CCCAAGCGGTTCG-5′

If the CDR is too long to incorporate all the desired mutations andremain under 70 nucleotides, the mutagenesis region may be broken upinto two regions. An example of this process is shown below, where the 8amino acid CDR as shown above is broken into two 4 amino acid regions(region 1 and region 2, respectively).

Region 1:

aa#                      1   2  3  4                         G   F  T  F5′-G TCTTTCTTGC GCTGCTTCCG GATTCACTTT CTCTAAGTAC TTTATGTTTT GGGTTC-3′(SEQ ID NO: 966)3′-C AGAAAGAACG CGACGAAGGC CTAAGTGAAA GAGATTCATG AAATACAAAA CCCAAG-5′(SEQ ID NO: 967)

Region 2:

aa#                       5  6  7   8                          S  K  Y   F5′-GCTGCTTCCG GATTCACTTT CTCTAAGTAC TTTATGTTTT GGGTTCGCCAAGC-3′(SEQ ID NO: 968)3′-CGACGAAGGC CTAAGTGAAA GAGATTCATG AAATACAAAA CCCAAGCGGTTCG-5′(SEQ ID NO: 969)

Sets of primers may be constructed to incorporate all 18 amino acidmutations at each position in region 2. Each codon selected for mutationmay be replaced with NHT, VAA or BGG in the sense direction. Exemplaryprimer sets for mutation of each of positions 5-8 are shown below.

Mutation of the S position (aa5) in region 2 above may be accomplishedby the following primers:R2-5-NHT5′-GCTGCTTCCGGATTCACTTT-CNHTAAGTACTTTATGTTTTGGGTTCGCCAAGC-3′(SEQ ID NO:970); R2-5-VAA5′-GCTGCTTCCGGATTCACTTTCVAAAAGTACTTTATGTTTTGGGTTCGCCAAGC-3′ (SEQ ID NO:971); and R2-5-BGG5′-GCTGCTTCCGGATTCACTTTCBGGAAGTAC-TTTATGT-TTTGGGTTCGCCAAGC-3′ (SEQ IDNO: 972).

Mutation of the K position (aa6) in region 2 above may be accomplishedby the following primers: R2-6-NHT5′-GCTGCTTCCGGATTCACTTTCTCTNHTTACTTTATGTTTTGGGTTCGCCAAGC-3′ (SEQ ID NO:973); R2-6-VAA5′-GCTGCTTCCGGATTCACTTTCTCTVAATACTTTATGTTTTGGGTTCGCCAAGC-3′ (SEQ ID NO:974); and R2-6-BGG 5′-GCTGCTTCCGGATTCACTTTCTCTBGGTACTTTATGTTTTGGGTTCGCCAAGC-3′ (SEQ ID NO: 975).

Mutation of the Y position (aa7) in region 2 above may be accomplishedby the following primers: R2-7-NHT 5′-GCTGCTTCCGGATTCACTTTCTCTAAGNHTTTTATGTTTTGGGTTCGCCAAGC-3′ (SEQ ID NO: 976); R2-7-VAA5′-GCTGCTTCCGGATTCACTTTCTCTAAGVAATTTATGTTTTGGGTTCGCCAAGC-3′ (SEQ ID NO:977); and R2-7-BGG 5′-GCTGCTTCCGGATTCACTTTCTCTAAGBGGTTTATGTTTTGGGTTCGCCAAGC-3′ (SEQ ID NO: 978).

Mutation of the F position (aa8) in region 2 above may be accomplishedby the following primers: R2-8-NHT 5′-GCTGCTTCCGGATTCACTTTCTCTAAGTACNHTATGTTTTGGGTTCGCCAAGC-3′ (SEQ ID NO: 979); R2-8-VAA5′-GCTGCTTCCGGATTCACTTTCTCTAAGTACVAAATGTTTTGGGTTCGCCAAGC-3′ (SEQ ID NO:980); and R2-8-BGG 5′-GCTGCTTCCGGATTCACTTTCTCTAAGTACBGGATGTTTTGGGTTCGCCAAGC-3′ (SEQ ID NO: 981).

Alternatively, modified antibody variable domains containing amino acidchanges at one or more contacting (C) residues present within anexemplary antibody may be synthesized by QUIKCHANGE™ site-directedmutagenesis (STRATAGENE, Texas).

In an exemplary method, QUIKCHANGE™ site-directed mutagenesis may beperformed to replace one or more codons in an antibody variable region(e.g., a CDR) such as XPA-23. Mutagenic primers are designed to containthe desired mutation and anneal to the same sequence on opposite strandsof a plasmid comprising a nucleotide coding for XPA-23. Preferably, thedesired mutation in the middle of the primer contains 20 bases ofcorrect sequence on both sides of the nucleic acid flanking themutation. The XPA-23 CDR1 coding region is shown below.

aa#                      1   2  3  4   5  6  7   8                         G   F  T  F   S  K  Y   F5′-G TCTTTCTTGC GCTGCTTCCG GATTCACTTT CTCTAAGTAC TTTATGTTTT(SEQ ID NO: 851) GGGTTCGCCAAGC-3′3′-C AGAAAGAACG CGACGAAGGC CTAAGTGAAA GAGATTCATG AAATACAAAA(SEQ ID NO: 852) CCCAAGCGGTTCG-5′

Primers for QUIKCHANGE™ site-directed mutagenesis are synthesized suchthat they are complementary to a parent nucleic acid sequence with theexception that they comprise a NHT, a VAA, or a BGG codon in the sensedirection, and a ADN, a TTB, or a CCV codon in the antisense directionat the position to be mutagenized in the parent nucleic acid. Exemplaryprimers for mutagenesis of each of the eight amino acid residues in theXPA-23 heavy chain CDR1 are shown below and comprise a degenerate codon(underlined nucleotide triplet):

Mutation of the G position (aa1) may be accomplished by the followingprimers: 5′-GTCTTTCTTGCGCTGCTTCCNHTTTCACTTTCTCTAAGTACTTTATG-3′ (SEQ IDNO: 853) and 3′-CAGAAAGAACGCGACGAAGGNDAAAGTGAAAGAGATTCATGAAATAC-5′ (SEQID NO: 854); 5′-GTCTTTCTTGCGCTGCTTCCVAATTCACTTTCTCTAAGTACTTTATG-3′ (SEQID NO: 855) and 3′-CAGAAAGAACGCGACGAAGGBTTAAGTGAAAGAGATTCATGAAATAC-5′(SEQ ID NO: 856); and5′-GTC-TTTCTTGCGCTGCTTCCBGGTTCACTTTCTCTAAGTACTTTATG-3′ (SEQ ID NO: 857)and 3′-CAGAAAGAACGCGACGAAGGVCCAAGTGAAAGAGATTCATGAAATAC-5′ (SEQ ID NO:858).

Mutation of the F position (aa2) may be accomplished by the followingprimers: 5′-CTTTCTTGCGCTGCTTCCGGANHTACTTTCTCTAAGTACTTTATG-3′ (SEQ ID NO:859) and 3′-GAAAGAACGCGACGAAGGCCTNDATGAAAGAGATTCATGAAATAC-5′ (SEQ ID NO:860); 5′-CTTTCTTGCGCTGCTTCCGGAVAAACTTTCTCTAAGTACTTTATG-3′ (SEQ ID NO:861) and 3′-GAAAGAACGCGACGAAGGCCTBTTTGAAAGAGATTCATGAAATAC-5′ (SEQ ID NO:862); and 5′-CTTTCTTGCGCTGCTTCCGGABGGACTTTCTCTAAGTACTTTATG-3′ (SEQ IDNO: 863) and 3′-GAAAGAACGCGACGAAGGCCTVCCTGAAAGAGATTCATGAAATAC-5′ (SEQ IDNO: 864).

Mutation of the T (aa3) position may be accomplished by the followingprimers: 5′-CTTGCGCTGCTTCCGGATTCNHTTTCTCTAAGTACTTTATGTTTTG-3′ (SEQ IDNO: 865) and 3′-GAACGCGACGAAGGCCTAAGNDAAAGAGATTCATGAAATACAAAAC-5′ (SEQID NO: 866); 5′-CTTGCGCTGCTTCCGGATTCVAATTCTCTAAGTACTTTATGTTTTG-3′ (SEQID NO: 867) and 3′-GAACGCGACGAAGGCCTAAGBTTAAGAGATTCATGAAATACAAAAC-5′(SEQ ID NO: 868); and5′-CTTGCGCTGCTTCCGGATTCBGGTTCTCTAAGTACTTTATGTTTTG-3′ (SEQ ID NO: 869)and 3′-GAACGCGACGAAGGCCTAAGVCCAAGAGATTCATGAAATACAAAAC-5′ (SEQ ID NO:870).

Mutation of the F (aa4) position may be accomplished by the followingprimers: 5′-CGCTGCTTCCGGATTCACTNHTTCTAAGTACTTTATGTTTTGGG-3′ (SEQ ID NO:871) and 3′-GCGACGAAGGCCTAAGTGANDAAGATTCATGAAATACAAAACCC-5′ (SEQ ID NO:872); 5′-CGCTGCTTCCGGATTCACTVAATCTAAGTACTTTATGTTTTGGG-3′ (SEQ ID NO:873) and 3′-GCGACGAAGGCCTAAGTGABTTAGATTCATGAAATACAAAACCC-5′ (SEQ ID NO:874); and 5′-CGCTGCTTCCGGATTCACTBGGTCTAAGTACTTTATGTTTTGGG-3′ (SEQ ID NO:875) and 3′-GCGACGAAGGCCTAAGTGAVCCAGATTCATGAAATACAAAACCC-5′ (SEQ ID NO:876).

Mutation of the S (aa5) position may be accomplished by the followingprimers: 5′-CTGCTTCCGGATTCACTTTCNHTAAGTACTTTATGTTTTGGGTTCG-3′ (SEQ IDNO: 877) and 3′-GACGAAGGCCTAAGTGAAAGNDATTCATGAAATACAAAACCCAAGC-5′ (SEQID NO: 878); 5′-CTGCTTCCGGATTCACTTTCVAAAAGTACTTTATGTTTTGGGTTCG-3′(SEQ IDNO: 879) and 3′-GACGAAGGCCTAAGTGAAAGBTTTTCATGAAATACAAAACCCAAGC-5′(SEQ IDNO: 880); and 5′-CTGCTTCCGGATTCACTTTCBGGAAGTACTTTATGTTTTGGGTTCG-3′(SEQID NO: 881) and 3′-GACGAAGGCCTAAGTGAAAGVCCTTCATGAAATACAAAACCCAAGC-5′(SEQID NO: 882).

Mutation of the K (aa6) position may be accomplished by the followingprimers: 5′-CTTCCGGATTCACTTTCTCTNHTTACTTTATGTTTTGGGTTCGCC-3′(SEQ ID NO:883) and 3′-GAAGGCCTAAGTGAAAGAGANDAATGAAATACAAAACCCAAGCGG-5′(SEQ ID NO:884); 5′-CTTCCGGATTCACTTTCTCTVAATACTTTATGTTTTGGGTTCGCC-3′(SEQ ID NO:885) and 3′-GAAGGCCTAAGTGAAAGAGABTTATGAAATACAAAACCCAAGCGG-5′(SEQ ID NO:886); and 5′-CTTCCGGATTCACTTTCTCTBGGTACTTTATGTTTTGGGTTCGCC-3′(SEQ ID NO:887) and 3′-GAAGGCCTAAGTGAAAGAGAVCCATGAAATACAAAACCCAAGCGG-5′(SEQ ID NO:888).

Mutation of the Y (aa7) position may be accomplished by the followingprimers: 5′-CCGGATTCACTTTCTCTAAGNHTTTTATGTTTTGGGTTCGCCAAG-3′(SEQ ID NO:889) and 3′-GGCCTAAGTGAAAGAGATTCNDAAAATACAAAACCCAAGCGGTTC-5′(SEQ ID NO:890); 5′-CCGGATTCACTTTCTCTAAGVAATTTATGTTTTGGGTTCGCCMG-3′(SEQ ID NO: 891)and 3′-GGCCTAAGTGAAAGAGATTCBTTAAATACAAAACCCAAGCGGTTC-5′(SEQ ID NO: 892);and 5′-CCGGATTCACTTTCTCTAAGBGGTTTATGTTTTGGGTTCGCCAAG-3′(SEQ ID NO: 893)and 3′-GGCCTAAGTGAAAGAGATTCVCCAAATACAAAACCCAAGCGGTTC-5′(SEQ ID NO: 894).

Mutation of the F (aa8) position may be accomplished by the followingprimers: 5′-GGATTCACTTTCTCTAAGTACNHTATGTTTTGGGTTCGCCAAGC-3′ (SEQ ID NO:895) and 3′-CCTAAGTGAAAGAGATTCATGNDATACAAAACCCAAGCGGTTCG-5′ (SEQ ID NO:896); 5′-GGATTCACTTTCTCTAAGTACVAAATGTTTTGGGTTCGCCAAGC-3′ (SEQ ID NO:897) and 3′-CCTAAGTGAAAGAGATTCATGBTTTACAAAACCCAAGCGGTTCG-5′ (SEQ ID NO:898); and 5′-GGATTCACTTTCTCTAAGTACBGGATGTTTTGGGTTCGCCAAGC-3′ (SEQ ID NO:899) and 3′-CCTAAGTGAAAGAGATTCATGVCCTACAAAACCCAAGCGGTTCG-5′ (SEQ ID NO:900).

C. Synthesis of Full-Length Mutagenized Antibody

Full-length mutagenized antibodies may be produced by recombinant DNAtechnologies.

For the PCR-based method, a first PCR reaction (PCR1) is performed witha R2-rev primer and a 5′-Sfil primer, which incorporates a 5′ Sfilrestriction site into the amplified fragment. For each libraryoligonucleotide containing the mutations described above, the PCR2reaction is performed to create the DNA fragment incorporating theprimer mutation and the 3′ Sfil restriction site. For the mutations inregion 2, twelve PCR2 reactions will be performed with forward primersdenoted R2-5 through R2-8 above (denoted primer-F in PCR2 below). Thereverse primer for the mutagenic reaction will be 3′-Sfil. Anappropriate amount of the following reagents may be used for PCR1:PfuUltra buffer; dNTPs [10 μM], template (10 ng total), 5′-Sfil [25pmol], R2-rev [25 pmol], PfuUltra (2.5 U/μL), dH2O to 50 μL total. Anappropriate amount of the following reagents may be used for PCR2:PfuUltra buffer, dNTPs [10 μM], template (10 ng total), Primer-F [10pmol], 3′-Sfil [25 pmol], PfuUltra (2.5 U/μL), dH2O to 50 μL total. PCR1and PCR2 may be conduced according to standard protocols including aninitial denatural step, a number of cycles including a denaturation,annealing and extension step and a final extension step for appropriatetimes and temperatures.

A full-length antibody fragment may be produced by performing a separatereaction for each PCR2 product. For this step, an approximatelyequimolar amount of PCR product 1 and 2 is combined (e.g., 0.5microliters of each PCR is combined). An appropriate amount of thefollowing reagents may be used generation of a full-length antibodyfragment: PfuUltra buffer, dNTPs [10 μM], PCR1 product, PCR2 product,PfuUltra (2.5 U/μL), dH2O to 50 μL total. PCR may be conduced accordingto standard protocols including an initial denatural step, a number ofcycles including a denaturation, annealing and extension step forappropriate times and temperatures.

The full-length fragment may then be amplified by directly adding to theabove reaction an appropriate amount of the following reagents: PfuUltrabuffer, dNTPs [10 μM], 5′-Sfil [25 pmol], 3′-Sfil [25 pmol], PfuUltra(2.5 U/μL), dH2O to 50 μL total. PCR may be conduced according tostandard protocols including an initial denaturation step, a number ofcycles that comprise a denaturation, annealing and extension step forappropriate times and temperatures and a final extension step. The PCRproduct may be examined on an agarose gel to ensure that the amplifiedDNA segment is the correct length.

Next, a vector and the DNA inserts obtained from the above PCR aredigested with Sfil (NEB) according to the manufacturer's instructionsand gel purified. The DNA synthesized fragment may be cloned into apXOMA Fab or pXOMA Fab-gIII vector. Briefly, the DNA fragment ispurified by using the QIAGEN® PCR purification kit and sequentiallydigesting the fragment with Notl (NEW ENGLAND BIOLABS® Ipswich, Mass.)and Ascl (NEW ENGLAND BIOLABS® Ipswich, Mass.) (See, Methods inMolecular Biology, vol. 178: Antibody Phage Display: Methods andProtocols Edited by: P. M. O'Brien and R. Aitken, Humana Press,“Standard Protocols for the Construction of Fab Libraries, Clark, M. A.,39-58) (see, e.g., FIG. 6). Next, the vectors may be ligated with themutagenized insert using T4 Ligase (NEW ENGLAND BIOLABS® Ipswich, Mass.)and transformed into TG1 cells by electroporation.

Alternatively, for the DPN-based method, a double-stranded DNA (e.g.,dsDNA) vector with an antibody insert isolated from a dam+ host is usedas template for mutagenesis. DNA isolated from almost all E. colistrains is dam methylated and therefore susceptible to Dpnl digestion.Two synthetic oligonucleotide primers containing the desired mutationeach complementary to opposite strands of the vector, are extendedduring temperature cycling by DNA polymerase (e.g., PfuTurbo). PCRreactions may comprise an appropriate amount of PfuUltra buffer, dNTPs[10 mM] each dNTP, template (50 ng total), Primer-F [5 μM], Primer-R [5μM], PfuUltra (2.5 U/μL), DMSO, and dH2O up to 50 μL total and beconducted with the following cycling parameters: an initialdenaturation, subsequent cycles of denaturation, annealing and extensionand a final extension step. Incorporation of the mutagenesis primersgenerates a mutated plasmid containing staggered nicks. Followingtemperature cycling, the PCR product is treated with Dpnl and incubatedat an appropriate temperature (e.g., at 37° C. for 4-5 hours). The Dpnlendonuclease (target sequence: 5′-Gm6ATC-3′) is specific for methylatedand hemimethylated DNA and is used to digest the parental DNA templateand to select for mutation-containing synthesized DNA. The nicked vectorDNA containing the desired mutations is then transformed intosupercompetent cells (e.g., XL1-Blue).

D. Sequencing of Mutagenized Antibodies

A library of mutagenized antibodies may comprise each of 18 unique aminoacid mutations at each position mutated. To identify all possible uniquemutations an appropriate number of clones obtained from each degeneratecodon are analyzed. For example, the NHT codon encodes 12 amino acidssuch that 72 clones from this reaction are sequenced for each mutatedposition. The VAA codon encodes 3 amino acids such that 12 clones aresequenced from this reaction for each mutated position. The BGG codonencodes 3 amino acids such that 12 clones from this reaction aresequenced for each mutated position. Unique clones are rearrayed into96-well plates.

E. Expression of Mutagenized Antibodies

Mutagenized antibodies may be expressed. In an exemplary method,starting cultures may be produced by filling a plate (e.g., a 96 wellplate) with an appropriate growth media (e.g., 2YTAG (2YT+2% glucose+100μgs/ml Ampicillin) and inoculating the plate with glycerol stocks of themutagenized antibodies. The cultures are then grown overnight (e.g., inan ATR plate shaker incubator at 37° C. with shaking at 450 rpm). Next,plates are filled with an appropriate growth medium (e.g., 1.2 mL perwell of Superbroth+100 μgs.ml Ampicillin+0.2% glucose). The plates arethen Inoculated with an appropriate amount of the overnight culture(e.g., 25 μL of overnight culture). The cultures are then grown withincubation (e.g., ATR plate shaker incubator at 37° C.) and shaking(e.g., at 700 rpm until Abs600 nm=1.5). Expression in the cultures isthen induced (e.g., by adding 12 uL of 100 mM IPTG per well to get afinal concentration of 1 mM IPTG final) and incubated overnight (e.g.,in an ATR plate shaker incubator at 30° C. with shaking at 700 rpm).Next, the plates are spun (e.g., at 4000 rpm using Beckman Coulter tabletop centrifuge for 10 minutes) and the supernatant decanted. The cellsare then vortexed to disturb and loosen the pellet. The pellets areresuspended (e.g., with 75 μL per well of cold PPB) and incubated oneice (e.g., for 10 minutes). Next, water (e.g., 225 μL per well) is addedand the cells resuspended. The suspension is incubated on ice (e.g., for1 hour) and the plates are then spun (e.g., at 4000 rpm using BeckmanCoulter table top centrifuge for 20 minutes). Last, the supernatants arecollected for use in assays as described in detail below.

F. ELISA Screening of Mutagenized Antibodies

An assay including, for example, an ELISA may be performed to ensurethat the mutagenized antibodies are capable of binding to theirrespective antigen.

In an exemplary ELISA, plates (e.g., 96-well Nunc Maxisorp plates) arecoated with an antibody to the mutagenized antibody (e.g., 50 μL perwell of 1 μg/ml Goat anti Human IgG (Fab)₂ Jackson immunoresearch, Cat.109-005-006) and the plates are then incubated overnight at 4° C. Afterincubation, the plates may be washed (e.g., 3× with PBS-Tween at 350μL/well) and then blocked (e.g., by adding 350 μL/well with 5%Milk+PBS).

Next, periplasmic extracts (PPE) containing the mutagenized antibody areblocked (e.g., by milk(diluted in PBS) to 200 μL of PPE to get a finalmilk percent of 5%). The PPEs are then mixed and incubated (e.g., atroom temperature still for 1 hour) before using as samples to screen onELISA and then washed (e.g., 3× with PBS-Tween at 350 μL/well). Theblocked PPE samples (e.g., 50 μL) are then added to the blocked ELISAplates and incubated (e.g., at room temperature for 1-2 hours). Againthe PPEs are washed (e.g., 3× with PBS-Tween at 350 μL/well). Next, anantibody specific for the mutagenized antibody is added to the PPEs(e.g., 50 μL/well of 1 μg/ml monoclonal anti-V5 antibody, SigmaCat.#V8012-50UG) and the PPEs incubated (e.g., at room temperature for 1hour). Again the PPEs are washed (e.g., 3× with PBS-Tween at 350μL/well). Next, a secondary antibody conjugated to a enzymatic label isadded to the PPEs (e.g., 1:10000 diluted Goat anti mouse HRP conjugated,Biorad, Cat. 170-5047) and incubated with the PPEs (e.g., for 1 hour atroom temperature). Again the PPEs are washed (e.g., 3× with PBS-Tween at350 μL/well). Next, an appropriate amount of substrate for the enzymaticlabel is added to the PPEs (e.g., 50 μL/well of TMB, soluble,Calbiochem, Cat. 613544) and the enzyme is allowed time to act on thesubstrate (e.g., until sufficiently blue color develops). The reactionmay be stopped by the addition of an agent that sequesters the substrateand/or and agent that inhibits the enzymatic activity of the secondaryantibody (e.g., 50 μL per well of 2N H₂SO₄). Last, absorbance of thesamples are read at 450 nm.

G. Ranking of Mutagenized Antibodies

Mutagenized antibodies may be ranked based on their dissociation ratefrom their respective antigen.

In an exemplary method, a Biacore A100 screening protocol may be used torank mutagenized antibody clones. For example, a CM5 chip may be dockedand normalized using normalization solution (e.g., using A100normalization solution and use and an appropriate running buffer (e.g.,HBS-N (0.01 M HEPES pH 7.4, 0.15 M NaCl). After normalization, softwareis set to immobilize antigen on desired spots of each flow cell. Forantigen surface preparation the surface may be activated (e.g., withNHS/EDC mixture from the amine coupling kit for 5 minutes at 10 μl/min).Antigen is then diluted (e.g., in 10 mM sodium acetate buffer) and thesurface of the CM5 chip is blocked (e.g., with 1 M ethanolamine HCl pH8.5 for 5 min at 10 μl/min). Next, each sample comprising a mutagenizedantibody is injected over the CM5 chip (e.g., for 3 min at 30 μl/minflow rate with 600 s dissociation) at an appropriate temperature (e.g.,25° C.). Biaevaluation software (e.g., Biacore A100 evaluation software)is then used to calculate dissociation rates of individual samples andthe relative amount of sample bound to each test surface. The data isfit to an appropriate kinetic model (e.g., the kinetic titration model).

Embodiments

1. A method for enhancing the binding affinity of a variable domain ofan antibody to a binding partner, to obtain a modified variable domainwith enhanced binding affinity to the binding partner, the methodcomprising:

-   -   a. identifying the proximity assigned to amino acid positions in        the variable domain of the antibody using the “prox” line as        shown in FIGS. 3A or 3B;    -   b. substituting one or more contacting (C) amino acid residues        with other amino acid residues, thereby generating a library of        modified variable domains;    -   c. screening the library for binding affinity to the binding        partner; and    -   d. obtaining a modified variable domain with enhanced binding        affinity to the binding partner.

2. The method of embodiment 1, wherein each contacting (C) residue issubstituted.

3. A method for enhancing the binding affinity of a variable domain ofan antibody to a binding partner, to obtain a modified variable domainwith enhanced binding affinity to the binding partner, the methodcomprising:

-   -   a. identifying the proximity assigned to amino acid positions in        the variable domain of the antibody using the “prox” line as        shown in FIGS. 3A or 3B;    -   b. substituting one or more peripheral (P) amino acid residues        with other amino acid residues, thereby generating a library of        modified variable domains;    -   c. screening the library for binding affinity to the binding        partner; and    -   d. obtaining a modified variable domain with enhanced binding        affinity to the binding partner.

4. The method of embodiment 3, wherein each peripheral (P) residue issubstituted.

5. A method for enhancing the binding affinity of a variable domain ofan antibody to a binding partner, to obtain a modified variable domainwith enhanced binding affinity to the binding partner, the methodcomprising:

-   -   a. identifying the proximity assigned to amino acid positions in        the variable domain of the antibody using the “prox” line as        shown in FIGS. 3A or 3B;    -   b. substituting one or more supporting (S) amino acid residues        with other amino acid residues, thereby generating a library of        modified variable domains;    -   c. screening        the-library-for-binding-affinity-to-the-binding-partner; and    -   d. obtaining a modified variable domain with enhanced binding        affinity to the binding partner.

6. The method of embodiment 5, wherein each supporting (S) residue issubstituted.

7. A method for enhancing the binding affinity of a variable domain ofan antibody to a binding partner, to obtain a modified variable domainwith enhanced binding affinity to the binding partner, the methodcomprising:

-   -   a. identifying the proximity assigned to amino acid positions in        the variable domain of the antibody using the “prox” line as        shown in FIGS. 3A or 3B;    -   b. substituting one or more interfacial (I) amino acid residues        with other amino acid residues, thereby generating a library of        modified variable domains;    -   c. screening the library for binding affinity to the binding        partner; and    -   d. obtaining a modified variable domain with enhanced binding        affinity to the binding partner.

8. The method of embodiment 7, wherein each interfacial (I) residue issubstituted.

9. The method of embodiment 1, wherein the contacting residue is incomplementarity determining domain-1 (CDR1) in a light chain variabledomain.

10. The method of embodiment 9, wherein the contacting residue is atposition 28, 30 or 31 in CDR1.

11. The method of embodiment 1, wherein the contacting residue is inCDR2 in a light chain variable domain.

12. The method of embodiment 11, wherein the contacting residue is atposition 50, 51 or 53 in CDR2.

13. The method of embodiment 1, wherein the contacting residue is inCDR1 in a heavy chain variable domain.

14. The method of embodiment 13, wherein the contacting residue is atposition 32 or 33 in CDR1.

15. The method of embodiment 1, wherein the contacting residue is inCDR2 in a heavy chain variable domain.

16. The method of embodiment 15, wherein the contacting residue is atposition 50, 52, 53, 54, 56, or 58 in CDR2.

17. The method of any one of embodiments 1, 3, 5 or 7, wherein the otheramino acid residues are alanine, arginine, asparagine, aspartic acid,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosineor valine.

18. The method of any one of embodiments 1, 3, 5 or 7, wherein the otheramino acid substitutions are introduced by PCR mutagenesis using primerswhich comprise one of seven degenerate codons.

19. The method of any one of embodiments 1, 3, 5 or 7, wherein thedegenerate codons are ARG (R=A/G), WMC (W=A/T; M=A/C), CAS (S=C/G), GAS(S=C/G), NTC (N=A/G/C/T), KGG (K=G/T) and SCG (S=C/G).

20. The method of any one of claim 1, 3, 5 or 7, wherein the degeneratecodons are NHT or NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (whereV=A/C/G) and BGG or DGG (where B=C/G/T, D=A/G/T).

21. The method of any one of embodiments 1, 3, 5 or 7, wherein thevariable domain is from a humanized antibody.

22. The method of any one of embodiments 1, 3, 5 or 7, wherein thevariable domain is from a human antibody.

23. The method of any one of embodiments 1, 3, 5 or 7, wherein bindingaffinity is determined by measuring K_(off).

24. A method of making a modified variable domain of an antibody withenhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. modifying the nucleotide sequence of an antibody variable        domain at one or more positions that encode a contacting (C)        residue identified from the “prox” line as shown in FIGS. 3A or        3B to produce amino acid substitutions at the C residue thereby        generating a library of modified antibody variable domains; and    -   b. selecting a modified variable domain from the library that        has enhanced binding affinity to the binding partner compared to        the parent variable domain.

25. The method of embodiment 24, wherein each contacting (C) residue issubstituted.

26. A method of making a modified variable domain of an antibody withenhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. modifying the nucleotide sequence of an antibody variable        domain at one or more positions that encode a peripheral (P)        residue identified from the “prox” line as shown in FIGS. 3A or        3B to produce amino acid substitutions at the P residue, thereby        generating a library of modified antibody variable domains; and    -   b. selecting a modified variable domain from the library that        has enhanced binding affinity to the binding partner compared to        the parent variable domain.

27. The method of embodiment 1, wherein each peripheral (P) residue issubstituted.

28. A method of making a modified variable domain of an antibody withenhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. modifying the nucleotide sequence of an antibody variable        domain at one or more positions that encode a supporting (S)        residue identified from the “prox” line as shown in FIGS. 3A or        3B to produce amino acid substitutions at the S residue thereby        generating a library of modified antibody variable domains; and    -   b. selecting a modified variable domain from the library that        has enhanced binding affinity to the binding partner compared to        the parent variable domain.

29. The method of embodiment 28, wherein each supporting (S) residue issubstituted.

30. A method of making a modified variable domain of an antibody withenhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. modifying the nucleotide sequence of an antibody variable        domain at one or more positions that encode an interfacial (I)        residue identified from the “prox” line as shown in FIGS. 3A or        3B to produce amino acid substitutions at the I residue, thereby        generating a library of modified antibody variable domains; and    -   b. selecting the modified variable domain from the library that        has enhanced binding affinity to a binding partner compared to a        parent variable domain compared to the parent variable domain.

31. The method of embodiment 30, wherein each interfacial (I) residue issubstituted.

32. The method of embodiment 24, wherein the contacting residue is incomplementarity determining domain-1 (CDR1) in a light chain variabledomain.

33. The method of embodiment 32, wherein the contacting residue is atposition 28, 30 or 31 in CDR1.

34. The method of embodiment 24 wherein the contacting residue is inCDR2 in a light chain variable domain.

35. The method of embodiment 34, wherein the contacting residue is atposition 50, 51 or 53 in CDR2.

36. The method of embodiment 24, wherein the contacting residue is inCDR1 in a heavy chain variable domain.

37. The method of embodiment 36, wherein the contacting residue is atposition 32 or 33 in CDR1.

38. The method of embodiment 24, wherein the contacting residue is inCDR2 in a heavy chain variable domain.

39. The method of embodiment 38, wherein the contacting residue is atposition 50, 52, 53, 54, 56, or 58 in CDR2.

40. The method of any one of embodiments 24, 26, 28 or 30 furthercomprising inserting the modified antibody variable domain into anappropriate vector.

41. The method of embodiment 40, wherein the vector is either a plasmidor a phage.

42. The method of any one of embodiment 41, wherein the vector is pXOMAFab or pXOMA Fab-gIII.

43. The method of any one of embodiments 24, 26, 28 or 30, wherein theamino acid substitutions are alanine, arginine, asparagine, asparticacid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosineor valine.

44. The method of any one of embodiments 24, 26, 28 or 30, wherein theamino acid substitutions are introduced by PCR mutagenesis using primerswhich comprise one of seven degenerate codons.

45. The method of any one of embodiments 24, 26, 28 or 30, wherein thedegenerate codons are ARG (R=A/G), WMC (W=A/T; M=A/C), CAS (S=C/G), GAS(S=C/G), NTC (N=A/G/C/T), KGG (K=G/T) and SCG (S=C/G).

46. The method of any one of embodiments 24, 26, 28 or 30, wherein thedegenerate codons are NHT or NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA(where V=A/C/G) and BGG or DGG (where B=C/G/T, D=A/G/T).

47. The method of any one of embodiments 24, 26, 28 or 30, wherein thevariable domain is from a humanized antibody.

48. The method of any one of embodiments 24, 26, 28 or 30, wherein thevariable domain is from a human antibody.

49. The method of any one of embodiments 24, 26, 28 or 30, whereinbinding affinity is determined by measuring K_(off).

50. The method of any one of embodiments 24, 26, 28 or 30, wherein step(b) comprises:

-   -   a. contacting a parent variable domain with the binding partner        under conditions that permit binding;    -   b. contacting the modified variable domains with binding partner        under conditions that permit binding; and    -   c. determining binding affinity of the modified variable domains        and the parent variable domain for the binding partner,

wherein modified variable domains that have a binding affinity for thebinding partner greater than the binding affinity of the parent variabledomain for the binding partner are identified as having enhanced bindingaffinity for the binding partner.

51. A method for selecting a modified variable domain of an antibodywith enhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. obtaining a library of modified antibody variable domains        comprising amino acid substitutions at a contacting (C) residue        identified from the “prox” line as shown in FIGS. 3A or 3B;    -   b. determining the binding affinity of the modified antibody        variable domains and the parent variable domain to the binding        partner; and    -   c. selecting the modified antibody variable domains that have        enhanced binding affinity to the binding partner compared to the        parent variable domain.

52. A method for selecting a modified variable domain of an antibodywith enhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. obtaining a library of modified antibody variable domains        comprising amino acid substitutions at a peripheral (P) residue        identified from the “prox” line as shown in FIGS. 3A or 3B;    -   b. determining the binding affinity of the modified antibody        variable domains and the parent variable domain to the binding        partner; and    -   c. selecting the modified antibody variable domains that have        enhanced binding affinity to the binding partner compared to the        parent variable domain.

53. A method for selecting a modified variable domain of an antibodywith enhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. obtaining a library of modified antibody variable domains        comprising amino acid substitutions at a supporting (S) residue        identified from the “prox” line as shown in FIGS. 3A or 3B;    -   b. determining the binding affinity of the modified antibody        variable domains and the parent variable domain to the binding        partner; and    -   c. selecting the modified antibody variable domains that have        enhanced binding affinity to the binding partner compared to the        parent variable domain.

54. A method for selecting a modified variable domain of an antibodywith enhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. obtaining a library of modified antibody variable domains        comprising amino acid substitutions at an interfacial (I)        residue identified from the “prox” line as shown in FIGS. 3A or        3B;    -   b. determining the binding affinity of the modified antibody        variable domains and the parent variable domain to the binding        partner; and    -   c. selecting the modified antibody variable domains that have        enhanced binding affinity to the binding partner compared to the        parent variable domain.

55. The method of embodiment 51, wherein the contacting residue is incomplementarity determining domain-1 (CDR1) in a light chain variabledomain.

56. The method of embodiment 55, wherein the contacting residue is atposition 28, 30 or 31 in CDR1.

57. The method of embodiment 51, wherein the contacting residue is inCDR2 in a light chain variable domain.

58. The method of embodiment 57, wherein the contacting residue is atposition 50, 51 or 53 in CDR2.

59. The method of embodiment 51, wherein the contacting residue is inCDR1 in a heavy chain variable domain.

60. The method of embodiment 59, wherein the contacting residue is atposition 32 or 33 in CDR1.

61. The method of embodiment 51, wherein the contacting residue is inCDR2 in a heavy chain variable domain.

62. The method of embodiment 61, wherein the contacting residue is atposition 50, 52, 53, 54, 56, or 58 in CDR2.

63. The method of any one of embodiments 51 to 54, wherein the aminoacid substitutions are alanine, arginine, asparagine, aspartic acid,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosineor valine.

64. The method of any one of embodiments 51 to 54, wherein the aminoacid substitutions are introduced by PCR mutagenesis using primers whichcomprise one of seven degenerate codons.

65. The method of any one of embodiments 51 to 54, wherein thedegenerate codons are ARG (R=A/G), WMC (W=A/T; M=A/C), CAS (S=C/G), GAS(S=C/G), NTC (N=A/G/C/T), KGG (K=G/T) and SCG (S=C/G).

66. The method of any one of embodiments 51 to 54, wherein thedegenerate codons are NHT or NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA(where V=A/C/G) and BGG or DGG (where B=C/GfT, D=A/G/T).

67. The method of any one of embodiments 51 to 54, wherein the variabledomain is from a humanized antibody.

68. The method of any one of embodiments 51 to 54, wherein the variabledomain is from a human antibody.

69. The method of any one of embodiments 51 to 54, wherein bindingaffinity is determined by measuring K_(off).

70. A method of producing a nucleic acid library with an equalrepresentation of one or more non-redundant amino acid changes at eachof one or more positions in a parent nucleic acid, the methodcomprising:

-   -   a. providing a set of primers that each comprise at least one        degenerate codon at identical positions, wherein the primers are        complementary to a sequence in the parent nucleic acid and the        primers code for an equal representation of non-redundant amino        acid changes at one or more positions;    -   b. hybridizing a primer from the set to the parent nucleic acid;    -   c. amplifying the parent nucleic acid molecule with the primer        to generate one or more nucleic acids that code for amino acid        changes at one or more identical positions;    -   d. repeating steps (b) and (c) with remaining primers from the        set;    -   e. pooling the nucleic acids produced with each primer in step        (d); and    -   f. obtaining a library of nucleic acids coding for an equal        representation of one or more amino acid changes at one or more        identical positions, with the proviso that the degenerate codons        do not code for methionine or cysteine.

71. The method of embodiment 70, wherein the primer set codes foreighteen amino acid changes at each of one or more positions in theparent nucleic acid.

72. The method of embodiment 71, wherein the set of primers comprisesthree primers.

73. The method of embodiment 71, wherein the set of primers comprisesseven primers.

74. The method of embodiment 72 or 73, wherein the primers each comprisea degenerate codon which collectively code for alanine, arginine,asparagine, aspartic acid, glutamine, glutamine acid, glycine,histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine and valine at each position.

75. The method of embodiment 72, wherein the primers each comprise oneor more degenerate codons as represented by NHT or NHC (where N=A/G/C/T,H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/G/T,D=A/G/T).

76. The method of embodiment 73, wherein the primers each comprise oneor more degenerate codons as represented by ARG (where R=A/G), WMC(where W=A/T and M=A/C), CAS (where S=C/G), GAS (where S=C/G), NTC(where N=A/G/C/T), KGG (where K=G/T) and SCG (where S=C/G).

77. The method of embodiment 70, wherein the primer set codes for basicamino acid changes at each of one or more positions in the parentnucleic acid.

78. The method of embodiment 77, wherein the primer set comprises oneprimer.

79. The method of embodiment 78, wherein the one primer comprises adegenerate codon which codes for arginine and lysine.

80. The method of embodiment 79, wherein the one primer comprises one ormore degenerate codons as represented by ARG (where, R=A/G).

81. The method of embodiment 70, wherein the primer set codes for polaramino acid changes at each of one or more positions in the parentnucleic acid.

82. The method of embodiment 81, wherein the primer set comprises twoprimers.

83. The method of embodiment 82, wherein the two primers each comprise adegenerate codon which collectively code for serine, threonine,asparagine and tyrosine.

84. The method of embodiment 83, wherein the two primers each compriseone or more degenerate codons as represented by WMC (where, W=A/T;M=A/C) and CAS (where S=C/G).

85. The method of embodiment 70, wherein the primer set codes for acidicamino acid changes at each of one or more positions in the parentnucleic acid.

86. The method of embodiment 85, wherein the primer set comprises onedegenerate codon.

87. The method of embodiment 86, wherein the one primer comprises adegenerate codon that codes for glutamic acid and aspartic acid.

88. The method of embodiment 87, wherein the one primer comprises one ormore degenerate codons as represented by GAS (where S=C/G).

89. The method of embodiment 70, wherein the primers code for non-polaramino acid changes at each of one or more positions in the parentnucleic acid.

90. The method of embodiment 89, wherein the primer set comprises threedegenerate codons.

91. The method of embodiment 90, wherein the three primers each comprisea degenerate codon that collectively code for glutamic acid and asparticacid.

92. The method of embodiment 91, wherein the primers each comprise oneor more degenerate codons as represented by NTC (where, N=A/G/C/T), KGG(where, K=G/T), and SCG (where S=C/G).

93. The method of embodiment 70, where the parent nucleic acid encodesan antibody variable region.

94. The method of embodiment 70, wherein the positions in the parentnucleic acid code for contacting (C) residues.

95. A set of primers comprising:

-   -   at least one degenerate codon at identical positions, wherein        the degenerate codons code for an equal representation of one or        more non-redundant amino acid changes at each of one or more        positions in the parent nucleic acid and the primers are        complementary to a sequence in the parent nucleic acid, with the        proviso that the degenerate codons do not code for methionine or        cysteine.

96. The set of primers of embodiment 95, wherein the primer set codesfor eighteen amino acid changes at each of one or more positions in theparent nucleic acid.

97. The set of primers of embodiment 96, wherein the set of primerscomprises three primers.

98. The set of primers of embodiment 96, wherein the set of primerscomprises seven primers.

99. The set of primers of embodiment 97 or 98, wherein the primers eachcomprise a degenerate codon which collectively code for alanine,arginine, asparagine, aspartic acid, glutamine, glutamine acid, glycine,histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine and valine at each position.

100. The method of embodiment 97, wherein the primers each comprise oneor more degenerate codons as represented by NHT or NHC (where N=A/G/C/T,H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/G/T,D=A/G/T).

101. The set of primers of embodiment 98, wherein the primers eachcomprise one or more degenerate codons as represented by ARG (whereR=A/G), WMC (where W=A/T and M=A/C), CAS (where S=C/G), GAS (whereS=C/G), NTC (where N=A/G/C/T), KGG (where K=G/T) and SCG (where S=C/G).

102. The set of primers embodiment 95, wherein the primer set codes forbasic amino acid changes at each of one or more positions in the parentnucleic acid.

103. The set of primers of embodiment 102, wherein the primer setcomprises one primer.

104. The set of primers of embodiment 103, wherein the one primercomprises a degenerate codon which codes for arginine and lysine.

105. The set of primers of embodiment 104, wherein the one primercomprises one or more degenerate codons as represented by ARG (where,R=A/G).

106. The set of primers of embodiment 95, wherein the primer set codesfor polar amino acid changes at each of one or more positions in theparent nucleic acid.

107. The set of primers of embodiment 106, wherein the primer setcomprises two primers.

108. The set of primers of embodiment 107, wherein the two primers eachcomprise a degenerate codon which collectively code for serine,threonine, asparagine and tyrosine.

109. The set of primers of embodiment 108, wherein the two primers eachcomprise one or more degenerate codons as represented by WMC (where,W=A/T; M=A/C) and CAS (where S=C/G).

110. The set of primers of embodiment 95, wherein the primer set codesfor acidic amino acid changes at each of one or more positions in theparent nucleic acid.

111. The set of primers of embodiment 110, wherein the primer setcomprises one degenerate codon.

112. The set of primers of embodiment 111, wherein the one primercomprises a degenerate codon that codes for glutamic acid and asparticacid.

113. The set of primers of embodiment 112, wherein the one primercomprises one or more degenerate codons as represented by GAS (whereS=C/G).

114. The set of primers of embodiment 95, wherein the primers code fornon-polar amino acid changes at each of one or more positions in theparent nucleic acid.

115. The set of primers of embodiment 114, wherein the primer setcomprises three degenerate codons.

116. The set of primers of embodiment 115, wherein the three primerseach comprise a degenerate codon that collectively code for glutamicacid and aspartic acid.

117. The set of primers of embodiment 116, wherein the primers eachcomprise one or more degenerate codons as represented by NTC (where,N=A/G/C/T), KGG (where, K=G/T), and SCG (where S=C/G).

118. The set of primers of embodiment 95, where the parent nucleic acidencodes an antibody variable region.

119. The set of primers of embodiment 95, wherein the positions in theparent nucleic acid code for contacting (C) residues.

120. A kit for mutagenesis of one or more positions in a parent nucleicacid, the kit comprising:

-   -   a set of primers comprising at least one degenerate codon at        identical positions, wherein the degenerate codons code for an        equal representation of one or more non-redundant amino acid        changes at each of one or more positions in the parent nucleic        acid and the primers are complementary to a sequence in the        parent nucleic acid,    -   with the proviso that the degenerate codons do not code for        methionine or cysteine.

121. The kit of embodiment 121, wherein the primer set codes foreighteen amino acid changes at each of one or more positions in theparent nucleic acid.

122. The kit of embodiment 121, wherein the set of primers comprisesthree primers.

123. The kit of embodiment 121, wherein the set of primers comprisesseven primers.

124. The kit of embodiment 122 or 123, wherein the primers each comprisea degenerate codon which collectively code for alanine, arginine,asparagine, aspartic acid, glutamine, glutamine acid, glycine,histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine and valine at each position.

125. The kit of embodiment 122, wherein the primers each comprise one ormore degenerate codons as represented by NHT or NHC (where N=A/G/C/T,H=A/C/T), VAG or VAA (where V=A/C/G) and BGG or DGG (where B=C/G/T,D=A/G/T).

126. The kit of embodiment 123, wherein the primers each comprise one ormore degenerate codons as represented by ARG (where R=A/G), WMC (whereW=A/T and M=A/C), CAS (where S=C/G), GAS (where S=C/G), NTC (whereN=A/G/C/T), KGG (where K=G/T) and SCG (where S=C/G).

127. The kit of embodiment 120, wherein the primer set codes for basicamino acid changes at each of one or more positions in the parentnucleic acid.

128. The kit of embodiment 127, wherein the primer set comprises oneprimer.

129. The kit of embodiment 128, wherein the one primer comprises adegenerate codon which codes for arginine and lysine.

130. The kit of embodiment 129, wherein the one primer comprises one ormore degenerate codons as represented by ARG (where, R=A/G). 131. Thekit of embodiment 120, wherein the primer set codes for polar amino acidchanges at each of one or more positions in the parent nucleic acid.

132. The kit of embodiment 131, wherein the primer set comprises twoprimers.

133. The kit of embodiment 132, wherein the two primers each comprise adegenerate codon which collectively code for serine, threonine,asparagine and tyrosine.

134. The kit of embodiment 133, wherein the two primers each compriseone or more degenerate codons as represented by WMC (where, W=A/T;M=A/C) and CAS (where S=C/G).

135. The kit of embodiment 120, wherein the primer set codes for acidicamino acid changes at each of one or more positions in the parentnucleic acid.

136. The kit of embodiment 135, wherein the primer set comprises onedegenerate codon.

137. The kit of embodiment 136, wherein the one primer comprises adegenerate codon that codes for glutamic acid and aspartic acid.

138. The kit of embodiment 137, wherein the one primer comprises one ormore degenerate codons as represented by GAS (where S=C/G).

139. The kit of embodiment 120, wherein the primers code for non-polaramino acid changes at each of one or more positions in the parentnucleic acid.

140. The kit of embodiment 139, wherein the primer set comprises threedegenerate codons.

141. The kit of embodiment 140, wherein the three primers each comprisea degenerate codon that collectively code for glutamic acid and asparticacid.

142. The kit of embodiment 141, wherein the primers each comprise one ormore degenerate codons as represented by NTC (where, N=A/G/C/T), KGG(where, K=G/T), and SCG (where S=C/G).

143. The kit of embodiment 120, where the parent nucleic acid encodes anantibody variable region.

144. The kit of embodiment 120, wherein the positions in the parentnucleic acid code for contacting (C) residues.

145. A method for enhancing the binding affinity of a variable domain ofan antibody to a binding partner, to obtain a modified variable domainwith enhanced binding affinity to the binding partner, the methodcomprising:

-   -   a. identifying the proximity assigned to amino acid positions in        the variable domain of the antibody using the “prox” line as        shown in FIGS. 3A or 3B;    -   b. substituting one or more contacting (C), peripheral (P),        supporting (S) and interfacial (I) amino acid residues with        other amino acid residues, thereby generating a library of        modified variable domains;    -   c. screening the library for binding affinity to the binding        partner; and    -   d. obtaining a modified variable domain with enhanced binding        affinity to the binding partner.

146. The method of embodiment 145, wherein each contacting (C),peripheral (P), supporting (S) and interfacial (I) residue issubstituted.

147. A method of making a modified variable domain of an antibody withenhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. modifying the nucleotide sequence of an antibody variable        domain at one or more positions that encode a contacting (C),        peripheral (P), supporting (S) and interfacial (I) residue        identified from the “prox” line as shown in FIGS. 3A or 3B to        produce amino acid substitutions at the C residue thereby        generating a library of modified antibody variable domains; and    -   b. selecting a modified variable domain from the library that        has enhanced binding affinity to the binding partner compared to        the parent variable domain.

148. The method of embodiment 24, wherein each contacting (C),peripheral (P), supporting (S) and interfacial (I) residue issubstituted.

149. A method for selecting a modified variable domain of an antibodywith enhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. obtaining a library of modified antibody variable domains        comprising amino acid substitutions at one or more contacting        (C), peripheral (P), supporting (S) and interfacial (I) residues        identified from the “prox” line as shown in FIGS. 3A or 3B;    -   b. determining the binding affinity of the modified antibody        variable domains and the parent variable domain to the binding        partner; and    -   c. selecting the modified antibody variable domains that have        enhanced binding affinity to the binding partner compared to the        parent variable domain.

150. The method of embodiment 149, wherein each contacting (C),peripheral (P), supporting (S) and interfacial (I) residue issubstituted.

1A. A method for enhancing the binding affinity of a variable domain ofan antibody to a binding partner, to obtain modified variable domainswith enhanced binding affinity to the binding partner, the methodcomprising:

-   -   a. identifying the proximity assigned to amino acid positions in        the variable domain of the antibody using the “prox” line as        shown in FIGS. 3A or 3B;    -   b. substituting one or more contacting (C) amino acid residues        in the antibody variable domain with other amino acid residues        to generate an array of modified variable domains;    -   c. screening the array of modified variable domains for binding        affinity to the binding partner; and    -   d. obtaining modified variable domains with enhanced binding        affinity to the binding partner.

2A. The method of embodiment 1, wherein each contacting (C) residue inthe antibody variable domain is separately substituted.

3A. The method of embodiment 1, wherein one or more contacting (C)residues in the antibody variable domain are simultaneously substituted.

4A. The method of embodiment 1, wherein the contacting residue is incomplementarity determining domain-1 (CDR1) in a light chain variabledomain.

5A. The method of embodiment 4, wherein the contacting residue is atposition 28, 30 or 31 in CDR1.

6A. The method of embodiment 1, wherein the contacting residue is inCDR2 in a light chain variable domain.

7A. The method of embodiment 6, wherein the contacting residue is atposition 50, 51 or 53 in CDR2.

8A. The method of embodiment 1, wherein the contacting residue is inCDR1 in a heavy chain variable domain.

9A. The method of embodiment 8, wherein the contacting residue is atposition 32 or 33 in CDR1.

10A. The method of embodiment 1, wherein the contacting residue is inCDR2 in a heavy chain variable domain.

11A. The method of embodiment 10, wherein the contacting residue is atposition 50, 52, 53, 54, 56, or 58 in CDR2.

12A. A method for enhancing the binding affinity of a variable domain ofan antibody to a binding partner, to obtain modified variable domainswith enhanced binding affinity to the binding partner, the methodcomprising:

-   -   a. identifying the proximity assigned to amino acid positions in        the variable domain of the antibody using the “prox” line as        shown in FIGS. 3A or 3B;    -   b. substituting one or more peripheral (P) amino acid residues        in the antibody variable domain with other amino acid residues        to generate an array of modified variable domains;    -   c. screening the array of modified variable domains for binding        affinity to the binding partner; and    -   d. obtaining modified variable domains with enhanced binding        affinity to the binding partner.

13A. The method of embodiment 12, wherein each peripheral (P) residue inthe antibody variable domain is separately substituted.

14A. The method of embodiment 12, wherein one or more peripheral (P)residues in the antibody variable domain are simultaneously substituted.

15A. A method for enhancing the binding affinity of a variable domain ofan antibody to a binding partner, to obtain modified variable domainswith enhanced binding affinity to the binding partner, the methodcomprising:

-   -   a. identifying the proximity assigned to amino acid positions in        the variable domain of the antibody using the “prox” line as        shown in FIGS. 3A or 3B;    -   b. substituting one or more supporting (S) amino acid residues        in the antibody variable domain with other amino acid residues        to generate an array of modified variable domains;    -   c. screening the array of modified variable domains for binding        affinity to the binding partner; and    -   d. obtaining modified variable domains with enhanced binding        affinity to the binding partner.

16A. The method of embodiment 15, wherein each supporting (S) residue inthe antibody variable domain is separately substituted.

17A. The method of embodiment 15, wherein one or more supporting (S)residues in the antibody variable domain are simultaneously substituted.

18A. A method for enhancing the binding affinity of a variable domain ofan antibody to a binding partner, to obtain modified variable domainswith enhanced binding affinity to the binding partner, the methodcomprising:

-   -   a. identifying the proximity assigned to amino acid positions in        the variable domain of the antibody using the “prox” line as        shown in FIGS. 3A or 3B;    -   b. substituting one or more interfacial (I) amino acid residues        in the antibody variable domain with other amino acid residues        to generate an array of modified variable domains;    -   c. screening the array of modified variable domains for binding        affinity to the binding partner; and    -   d. obtaining modified variable domains with enhanced binding        affinity to the binding partner.

19A. The method of embodiment 1, wherein each interfacial (I) residue inthe antibody variable domain is separately substituted.

20A. The method of embodiment 1, wherein one or more interfacial (I)residues in the antibody variable domain are simultaneously substituted.

21A. The method of any one of embodiments 1, 12, 15 or 18, wherein theother amino acid residues are alanine, arginine, asparagine, asparticacid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosineor valine.

22A. The method of any one of embodiments 1, 12, 15 or 18, wherein theother amino acid substitutions are introduced by PCR mutagenesis usingprimers which comprise one of seven degenerate codons.

23A. The method of embodiment 22, wherein the degenerate codons are ARG(R=A/G), WMC (W=A/T; M=A/C), CAS (S=C/G), GAS (S=C/G), NTC (N=A/G/C/T),KGG (K=G/T) and SCG (S=C/G).

24A. The method of embodiment 22, wherein the degenerate codons are NHTor NHC (where N=A/G/C/T, H=NC/T), VAG or VAA (where V=A/C/G) and BGG orDGG (where B=C/G/T, D=A/G/T).

25A. The method of any one of embodiments 1, 12, 15 or 18, wherein thevariable domain is from a humanized antibody.

26A. The method of any one of embodiments 1, 12, 15 or 18, wherein thevariable domain is from a human antibody.

27A. The method of any one of embodiments 1, 12, 15 or 18, whereinbinding affinity is determined by measuring K_(off).

28A. The method of making modified variable domains of an antibody withenhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. modifying the nucleotide sequence of an antibody variable        domain at one or more positions that encode contacting (C)        residues identified from the “prox” line as shown in FIGS. 3A or        3B to produce amino acid substitutions at C residues to generate        an array of modified antibody variable domains; and    -   b. selecting modified variable domains from the array that have        enhanced binding affinity to the binding partner compared to the        parent variable domain.

29A. The method of embodiment 28, wherein each contacting (C) residue inthe antibody variable domain is separately substituted.

30A. The method of embodiment 28, wherein one or more contacting (C)residues in the antibody variable domain are simultaneously substituted.

31A. The method of embodiment 28, wherein the contacting residue is incomplementarity determining domain-1 (CDR1) in a light chain variabledomain.

32A. The method of embodiment 31, wherein the contacting residue is atposition 28, 30 or 31 in CDR1.

33A. The method of embodiment 28, wherein the contacting residue is inCDR2 in a light chain variable domain.

34A. The method of embodiment 33, wherein the contacting residue is atposition 50, 51 or 53 in CDR2.

35A. The method of embodiment 28, wherein the contacting residue is inCDR1 in a heavy chain variable domain.

36A. The method of embodiment 35, wherein the contacting residue is atposition 32 or 33 in CDR1.

37A. The method of embodiment 28, wherein the contacting residue is inCDR2 in a heavy chain variable domain.

38A. The method of embodiment 37, wherein the contacting residue is atposition 50, 52, 53, 54, 56, or 58 in CDR2.

39A. A method of making modified variable domains of an antibody withenhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. modifying the nucleotide sequence of an antibody variable        domain at one or more positions that encode peripheral (P)        residues identified from the “prox” line as shown in FIGS. 3A or        3B to produce amino acid substitutions at P residues to generate        an array of modified antibody variable domains; and    -   b. selecting modified variable domains from the array that have        enhanced binding affinity to the binding partner compared to the        parent variable domain.

40A. The method of embodiment 39, wherein each peripheral (P) residue inthe antibody variable domain is separately substituted.

41A. The method of embodiment 39, wherein one or more peripheral (P)residues in the antibody variable domain are simultaneously substituted.

42A. A method of making modified variable domains of an antibody withenhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. modifying the nucleotide sequence of an antibody variable        domain at one or more positions that encode supporting (S)        residues identified from the “prox” line as shown in FIGS. 3A or        3B to produce amino acid substitutions at S residues to generate        an array of modified antibody variable domains; and    -   b. selecting modified variable domains from the array that have        enhanced binding affinity to the binding partner compared to the        parent variable domain.

43A. The method of embodiment 42, wherein each supporting (S) residue inthe antibody variable domain is separately substituted.

44A. The method of embodiment 42, wherein one or more supporting (S)residues in the antibody variable domain are simultaneously substituted.

45A. A method of making modified variable domains of an antibody withenhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. modifying the nucleotide sequence of an antibody variable        domain at one or more positions that encode interfacial (I)        residues identified from the “prox” line as shown in FIGS. 3A or        3B to produce amino acid substitutions at I residues to generate        an array of modified antibody variable domains; and    -   b. selecting modified variable domains from the array that have        enhanced binding affinity to the binding partner compared to the        parent variable domain.

46A. The method of embodiment 45, wherein each interfacial (I) residuein the antibody variable domain is separately substituted.

47A. The method of embodiment 45, wherein one or more interfacial (I)residues in the antibody variable domain are simultaneously substituted.

48A. The method of any one of embodiments 28, 39, 42 or 45 furthercomprising inserting the modified antibody variable domains into anappropriate vector.

49A. The method of embodiment 48, wherein the vector is either a plasmidor a phage.

50A. The method of any one of embodiment 49, wherein the vector is pXOMAFab or pXOMA Fab-gIII.

51A. The method of any one of embodiments 28, 39, 42 or 45, wherein theamino acid substitutions are alanine, arginine, asparagine, asparticacid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosineor valine.

52A. The method of any one of embodiments 28, 39, 42 or 45, wherein theamino acid substitutions are introduced by PCR mutagenesis using primerswhich comprise one of seven degenerate codons.

53A. The method of embodiment 52, wherein the degenerate codons are ARG(R=A/G), WMC (W=A/T; M=A/C), CAS (S=C/G), GAS (S=C/G), NTC (N=A/G/C/T),KGG (K=G/T) and SCG (S=C/G).

54A. The method of embodiment 52, wherein the degenerate codons are NHTor NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG orDGG (where B=C/G/T, D=A/G/T).

55A. The method of any one of embodiments 28, 39, 42 or 45, wherein thevariable domain is from a humanized antibody.

56A. The method of any one of embodiments 28, 39, 42 or 45, wherein thevariable domain is from a human antibody.

57A. The method of any one of embodiments 28, 39, 42 or 45, whereinbinding affinity is determined by measuring K_(off).

58A. The method of any one of embodiments 28, 39, 42 or 45, wherein step(b) comprises:

-   -   a. contacting a parent variable domain with the binding partner        under conditions that permit binding;    -   b. contacting the modified variable domains with binding partner        under conditions that permit binding; and    -   c. determining binding affinity of the modified variable domains        and the parent variable domain for the binding partner,

wherein modified variable domains that have a binding affinity for thebinding partner greater than the binding affinity of the parent variabledomain for the binding partner are identified as having enhanced bindingaffinity for the binding partner.

59A. A method for selecting modified variable domains of an antibodywith enhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. obtaining an array of modified antibody variable domains        comprising amino acid substitutions at one or more        contacting (C) residues identified from the “prox” line as shown        in FIGS. 3A or 3B;    -   b. determining the binding affinity of the modified antibody        variable domains and the parent variable domain to the binding        partner; and    -   c. selecting the modified antibody variable domains that have        enhanced binding affinity to the binding partner compared to the        parent variable domain.

60A. A method for selecting modified variable domains of an antibodywith enhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. obtaining an array of modified antibody variable domains        comprising amino acid substitutions at one or more        peripheral (P) residues identified from the “prox” line as shown        in FIGS. 3A or 3B;    -   b. determining the binding affinity of the modified antibody        variable domains and the parent variable domain to the binding        partner; and    -   c. selecting the modified antibody variable domains that have        enhanced binding affinity to the binding partner compared to the        parent variable domain.

61A. A method for selecting modified variable domains of an antibodywith enhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. obtaining an array of modified antibody variable domains        comprising amino acid substitutions at one or more        supporting (S) residues identified from the “prox” line as shown        in FIGS. 3A or 3B;    -   b. determining the binding affinity of the modified antibody        variable domains and the parent variable domain to the binding        partner; and    -   c. selecting the modified antibody variable domains that have        enhanced binding affinity to the binding partner compared to the        parent variable domain.

62A. A method for selecting modified variable domains of an antibodywith enhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. obtaining an array of modified antibody variable domains        comprising amino acid substitutions at one or more        interfacial (I) residues identified from the “prox” line as        shown in FIGS. 3A or 3B;    -   b. determining the binding affinity of the modified antibody        variable domains and the parent variable domain to the binding        partner; and    -   c. selecting the modified antibody variable domains that have        enhanced binding affinity to the binding partner compared to the        parent variable domain.

63A. The method of embodiment 59, wherein the contacting residue is incomplementarity determining domain-1 (CDR1) in a light chain variabledomain.

64A. The method of embodiment 63, wherein the contacting residue is atposition 28, 30 or 31 in CDR1.

65A. The method of embodiment 59, wherein the contacting residue is inCDR2 in a light chain variable domain.

66A. The method of embodiment 65, wherein the contacting residue is atposition 50, 51 or 53 in CDR2.

67A. The method of embodiment 59, wherein the contacting residue is inCDR1 in a heavy chain variable domain.

68A. The method of embodiment 67, wherein the contacting residue is atposition 32 or 33 in CDR1.

69A. The method of embodiment 59, wherein the contacting residue is inCDR2 in a heavy chain variable domain.

70A. The method of embodiment 69, wherein the contacting residue is atposition 50, 52, 53, 54, 56, or 58 in CDR2.

71A. The method of any one of embodiments 59 to 62, wherein the aminoacid substitutions are alanine, arginine, asparagine, aspartic acid,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosineor valine.

72A. The method of any one of embodiments 59 to 62, wherein the aminoacid substitutions are introduced by PCR mutagenesis using primers whichcomprise one of seven degenerate codons.

73A. The method of embodiment 72, wherein the degenerate codons are ARG(R=A/G), WMC (W=A/T; M=A/C), CAS (S=C/G), GAS (S=C/G), NTC (N=A/G/C/T),KGG (K=G/T) and SCG (S=C/G).

74A. The method of embodiment 72, wherein the degenerate codons are NHTor NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA (where V=A/C/G) and BGG orDGG (where B=C/G/T, D=A/G/T).

75A. The method of any one of embodiments 59 to 62, wherein the variabledomain is from a humanized antibody.

76A. The method of any one of embodiments 59 to 62, wherein the variabledomain is from a human antibody.

77A. The method of any one of embodiments 59 to 62, wherein bindingaffinity is determined by measuring K_(off).

78A. A method for generating an array of modified antibody variabledomains with eighteen amino acid changes at one or more contacting (C)residues from a collection of modified variable domains, said methodcomprising:

-   -   a. obtaining a collection of modified antibody variable domains        containing amino acid changes at one or more contacting (C)        residues;    -   b. sequencing the collection of modified variable domains; and    -   c. arranging each sequenced modified antibody variable domain        comprising one of the eighteen amino acid changes at one or more        contacting (C) residue to generate an array of modified variable        domains with eighteen amino acid changes at one or more        contacting (C) residues.

79A. The method of embodiment 78, wherein the collection is a library.

80A. A method for generating an array of modified variable domains witheighteen amino acid changes at one or more contacting (C) residues, saidmethod comprising:

-   -   a. synthesizing polynucleotides that encode sequences that vary        at one or more contacting (C) residues and contain eighteen        amino acid changes at each contacting (C) residue to generate        modified antibody variable domains; and    -   b. arranging each synthesized polynucleotide from step (a) to        generate an array of synthesized polynucleotides with eighteen        amino acid changes at one or more contacting (C) residues.

81A. A method for generating an array of modified variable domains witheighteen amino acid changes at one or more contacting (C) residues, saidmethod comprising:

-   -   a. synthesizing polynucleotides that encode sequences that vary        at one or more contacting (C) residues and contain eighteen        amino acid changes at each contacting (C) residue to generate        modified antibody variable domains;    -   b. transfecting each synthesized polynucleotide of step (a)        separately into a host cell to generate clones comprising the        synthesized polynucleotides; and    -   c. arranging each clone from step (b) to generate an array of        clones capable of expressing modified variable domains with        eighteen amino acid changes at one or more contacting (C)        residues.

82A. The method of any one of embodiments 78, 80 or 81, wherein eachcontacting (C) residue in the antibody variable domain is separatelychanged.

83A. The method of any one of embodiments 78, 80 or 81, wherein one ormore contacting (C) residues in the antibody variable domain aresimultaneously changed.

84A. The method of any one of embodiments 78, 80 or 81, wherein thecontacting residue is in complementarity determining domain-1 (CDR1) ina light chain variable domain.

85A. The method of any one of embodiments 78, 80 or 81, wherein thecontacting residue is at position 28, 30 or 31 in CDR1.

86A. The method of any one of embodiments 78, 80 or 81, wherein thecontacting residue is in CDR2 in a light chain variable domain.

87A. The method of any one of embodiments 78, 80 or 81, wherein thecontacting residue is at position 50, 51 or 53 in CDR2.

88A. The method of any one of embodiments 78, 80 or 81, wherein thecontacting residue is in CDR1 in a heavy chain variable domain.

89A. The method of any one of embodiments 78, 80 or 81, wherein thecontacting residue is at position 32 or 33 in CDR1.

90A. The method of any one of embodiments 78, 80 or 81, wherein thecontacting residue is in CDR2 in a heavy chain variable domain.

91A. The method of any one of embodiments 78, 80 or 81, wherein thecontacting residue is at position 50, 52, 53, 54, 56, or 58 in CDR2.

92A. The method of any one of any one of embodiments 78, 80 or 81,wherein the amino acid changes are alanine, arginine, asparagine,aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine or valine.

93A. The method of any one of embodiments 78, 80 or 81, wherein theamino acid changes are introduced by PCR mutagenesis using primers whichcomprise one of seven degenerate codons.

94A. The method of any one of embodiments 78, 80 or 81, wherein thedegenerate codons are ARG (R=A/G), WMC (W=A/T; M=A/C), CAS (S=C/G), GAS(S=C/G), NTC (N=A/G/C/T), KGG (K=G/T) and SCG (S=C/G).

95A. The method of any one of embodiments 78, 80 or 81, wherein thedegenerate codons are NHT or NHC (where N=A/G/C/T, H=A/C/T), VAG or VAA(where V=A/C/G) and BGG or DGG (where B=C/G/T, D=A/G/T).

96A. The method of any one of embodiments 78, 80 or 81, wherein thevariable domain is from a humanized antibody.

97A. The method of any one of embodiments 78, 80 or 81, wherein thevariable domain is from a human antibody.

98A. A method for enhancing the binding affinity of a variable domain ofan antibody to a binding partner, to obtain modified variable domainswith enhanced binding affinity to the binding partner, the methodcomprising:

-   -   a. identifying the proximity assigned to amino acid positions in        the variable domain of the antibody using the “prox” line as        shown in FIGS. 3A or 3B;    -   b. substituting one or more contacting (C), peripheral (P),        supporting (S) and interfacial (I) amino acid residues in the        antibody variable domain with other amino acid residues to        generate an array of modified variable domains;    -   c. screening the array of modified variable domains for binding        affinity to the binding partner; and    -   d. obtaining modified variable domains with enhanced binding        affinity to the binding partner.

99A. The method of embodiment 98, wherein each contacting (C),peripheral (P), supporting (S) and interfacial (I) residue in theantibody variable domain is separately substituted.

100A. A method of making modified variable domains of an antibody withenhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. modifying the nucleotide sequence of an antibody variable        domain at one or more positions that encode contacting (C),        peripheral (P), supporting (S) and interfacial (I) residues        identified from the “prox” line as shown in FIGS. 3A or 3B to        produce amino acid substitutions at C residues to generate an        array of modified antibody variable domains; and    -   b. selecting modified variable domains from the array that have        enhanced binding affinity to the binding partner compared to the        parent variable domain.

101A. The method of embodiment100, wherein each contacting (C),peripheral (P), supporting (S) and interfacial (I) residue in theantibody variable domain is separately substituted.

102A. A method for selecting modified variable domains of an antibodywith enhanced binding affinity to a binding partner compared to a parentvariable domain, the method comprising:

-   -   a. obtaining an array of modified antibody variable domains        comprising amino acid substitutions at one or more contacting        (C), peripheral (P), supporting (S) and interfacial (I) residue        identified from the “prox” line as shown in FIGS. 3A or 3B;    -   b. determining the binding affinity of the modified antibody        variable domains and the parent variable domain to the binding        partner; and    -   c. selecting the modified antibody variable domains that have        enhanced binding affinity to the binding partner compared to the        parent variable domain.

103A. The method of embodiment 102, wherein each contacting (C),peripheral (P), supporting (S) and interfacial (I) residue in theantibody variable domain is separately substituted.

1B. An antibody comprising an ING-1 heavy chain variable region as setforth in SEQ ID NO: 579 comprising a substitution at position 28 or 30in HCDR1.

2B. The heavy chain variable region of embodiment 1B, wherein thesubstitution at position 28 is selected from the group consisting of:T28V, T28I and T28P.

3B. The heavy chain variable region of embodiment 1B, wherein thesubstitution at position 30 is T30Y.

4B. An antibody comprising an ING-1 heavy chain variable region as setforth in SEQ ID NO: 579 comprising a substitution at position 59 inHCDR2.

5B. The heavy chain variable region of embodiment 4B, wherein thesubstitution at position 59 is T59W.

6B. An antibody comprising an ING-1 heavy chain variable region as setforth in SEQ ID NO: 579 comprising a substitution at position 100, 101or 102 in HCDR3.

7B. The heavy chain variable region of embodiment 6B, wherein thesubstitution at position 100 is G100R.

8B. The heavy chain variable region of embodiment 6B, wherein thesubstitution at position 101 is selected from the group consisting of:S101K, S101Q, S101V, S101I, S101G.

9B. The heavy chain variable region of embodiment 6B, wherein thesubstitution at position 102 in HCDR3 is selected from the groupconsisting of: A102R, A102H, A102Y, A102W, A102F and A102G.

10B. An antibody comprising an ING-1 light chain variable region as setforth in SEQ ID NO: 580 comprising a substitution at position 28 or 29in LCDR1.

11B. The light chain variable region of embodiment 10B, wherein thesubstitution at position 28 in LCDR1 is selected from the groupconsisting of: S28R, S28K, S28H, S28Y, S28F, S28Q, S28V, S28I and S28L.

12B. The light chain variable region of embodiment 10B, wherein thesubstitution at position 29 in LCDR1 is selected from the groupconsisting of L29S and L29A.

13B. An antibody comprising an ING-1 light chain variable region as setforth in SEQ ID NO: 580 comprising a substitution at 54, 55 or 58 inLCDR2.

14B. The light chain variable region of embodiment 13B, wherein thesubstitution at position 54 in LCDR2 is selected from the groupconsisting of: Y54K and Y54L.

15B. The light chain variable region of embodiment 13B, wherein thesubstitution at position 55 in LCDR2 is selected from the groupconsisting of: Q55R, Q55H and Q55W.

16B. The light chain variable region of embodiment 13B, wherein thesubstitution at position 58 in LCDR2 is selected from the groupconsisting of: N58W, N58V, N58I and N58P.

17B. An antibody comprising an ING-1 light chain variable region as setforth in SEQ ID NO: 580 comprising a substitution at position 97, 98, 99or 100 in LCDR3.

18B. The light chain variable region of embodiment 17B, wherein thesubstitution at position 97 in LCDR3 is L97I.

19B. The light chain variable region of embodiment 17B, wherein thesubstitution at position 98 in LCDR3 is selected from the groupconsisting of: E98R, E98K, E98T, E98S and E98L.

20B. The light chain variable region of embodiment 17B, wherein thesubstitution at position 99 in LCDR3 is L99I.

21B. The light chain variable region of embodiment 17B, wherein thesubstitution at position 100 in LCDR3 is P100Y.

22B. An antibody comprising an XPA-23 light chain variable region as setforth in SEQ ID NO: 582 comprising a substitution at position 27, 28 or30 in LCDR1.

23B. The light chain variable region of embodiment 22B, wherein thesubstitution at position 27 in LCDR1 is selected from the groupconsisting of: Q27S, Q27F and Q27G.

24B. The light chain variable region of embodiment 22B, wherein thesubstitution at position 28 in LCDR1 is selected from the groupconsisting of: D28I, D28S and D28W.

25B. The light chain variable region of embodiment 22B, wherein thesubstitution at position 30 in LCDR1 is N30F.

26B. An antibody comprising an XPA-23 light chain variable region as setforth in SEQ ID NO: 582 comprising a substitution at position 51 or 53in LCDR2.

27B. The light chain variable region of embodiment 26B, wherein thesubstitution at position 51 in LCDR2 is A51 G.

28B. The light chain variable region of embodiment 26B, wherein thesubstitution at position 53 in LCDR2 is selected from the groupconsisting of: S53K and S53R.

29B. An antibody comprising an XPA-23 light chain variable region as setforth in SEQ ID NO: 582 comprising a substitution at position 92, 93, 95or 96 in LCDR3.

30B. The light chain variable region of embodiment 29B, wherein thesubstitution at position 92 in LCDR3 is D92S.

31B. The light chain variable region of embodiment 29B, wherein thesubstitution at position 93 in LCDR3 is selected from the groupconsisting of: S93D and S93E.

32B. The light chain variable region of embodiment 29B, wherein thesubstitution at position 95 in LCDR3 is selected from the groupconsisting of: P95S and P95A.

33B. The light chain variable region of embodiment 29B, wherein thesubstitution at position 96 in LCDR3 is L96W.

34B. An antibody comprising an XPA-23 heavy chain variable region as setforth in SEQ ID NO: 581 comprising a substitution at position 135, 138,139, 140 or 142 in HCDR1.

35B. The heavy chain variable region of embodiment 34B, wherein thesubstitution at position 135 in HCDR1 is selected from the groupconsisting of: T135K and T135E.

36B. The heavy chain variable region of embodiment 34B, wherein thesubstitution at position 138 in HCDR1 is selected from the groupconsisting of: K138Y, K138W, K138E, K138L, K138P and K138H.

37B. The heavy chain variable region of embodiment 34B, wherein thesubstitution at position 139 in HCDR1 is Y139H.

38B. The heavy chain variable region of embodiment 34B, wherein thesubstitution at position 140 in HCDR1 is F140I.

39B. The heavy chain variable region of embodiment 34B, wherein thesubstitution at position 142 in HCDR1 is selected from the groupconsisting of: F142T and F142A.

40B. An antibody comprising an XPA-23 heavy chain variable region as setforth in SEQ ID NO: 581 comprising a substitution at position 161 or 163in HCDR2.

41B. The heavy chain variable region of embodiment 40B, wherein thesubstitution at position 161 in HCDR2 is selected from the groupconsisting of: S161R and S161K.

42B. The heavy chain variable region of embodiment 40B, wherein thesubstitution at position 163 in HCDR2 is selected from the groupconsisting of: G163L, G163Q, G163W, G163Y, G163I, G163K, G163R andG163F.

43B. An antibody comprising an XPA-23 heavy chain variable region as setforth in SEQ ID NO: 581 comprising a substitution at position 208, 210,211 or 212 in HCDR3.

44B. The heavy chain variable region of embodiment 43B, wherein thesubstitution at position 208 in HCDR3 is Y208L.

45B. The heavy chain variable region of embodiment 43B, wherein thesubstitution at position 210 in HCDR3 is G210V.

46B. The heavy chain variable region of embodiment 43B, wherein thesubstitution at position 211 in HCDR3 is selected from the groupconsisting of: N211A and N211V.

47B. The heavy chain variable region of embodiment 43B, wherein thesubstitution at position 212 in HCDR3 is selected from the groupconsisting of: S212E and S212P.

While the present disclosure has been described and illustrated hereinby references to various specific materials, procedures and examples, itis understood that the disclosure is not restricted to the particularcombinations of materials and procedures selected for that purpose.Numerous variations of such details can be implied as will beappreciated by those skilled in the art. It is intended that thespecification and examples be considered as exemplary, only, with thetrue scope and spirit of the disclosure being indicated by the followingclaims. All references, patents, and patent applications referred to inthis application are herein incorporated by reference in their entirety.

1. A method for enhancing the binding affinity of a variable domain ofan antibody to a binding partner, to obtain a modified variable domainwith enhanced binding affinity to the binding partner, the methodcomprising: a. identifying the proximity assigned to amino acidpositions in the variable domain of the antibody using the “prox” lineas shown in FIGS. 3A, 3B, 3C or 3D; b. substituting one or morecontacting (C), peripheral (P), supporting (S), and/or interfacial (I)amino acid residues with other amino acid residues, thereby generating alibrary or an array of modified variable domains; c. screening thelibrary or the array for binding affinity to the binding partner; and d.obtaining a modified variable domain with enhanced binding affinity tothe binding partner. 2-3. (canceled)
 4. A method of making a modifiedvariable domain of an antibody with enhanced binding affinity to abinding partner compared to a parent variable domain, the methodcomprising: a. modifying the nucleotide sequence of an antibody variabledomain at one or more positions that encode a contacting (C), peripheral(P), supporting (S) and/or interfacial (I) residue identified from the“prox” line as shown in FIGS. 3A, 3B, 3C or 3D to produce amino acidsubstitutions at the C, P, S and/or I residue thereby generating alibrary or an array of modified antibody variable domains; and b.selecting a modified variable domain from the library or the array thathas enhanced binding affinity to the binding partner compared to theparent variable domain.
 5. (canceled)
 6. A method for selecting amodified variable domain of an antibody with enhanced binding affinityto a binding partner compared to a parent variable domain, the methodcomprising: a. obtaining a library or an array of modified antibodyvariable domains comprising amino acid substitutions at one or morecontacting (C), peripheral (P), supporting (S) and/or interfacial (I)residues identified from the “prox” line as shown in FIGS. 3A, 3B, 3C or3D; b. determining the binding affinity of the modified antibodyvariable domains and the parent variable domain to the binding partner;and c. selecting the modified antibody variable domains that haveenhanced binding affinity to the binding partner compared to the parentvariable domain. 7-31. (canceled)
 32. A method of mutagenesis of aparent nucleic acid encoding an antibody variable domain to generatemodified antibody variable domains, said method comprising: (a.)obtaining one or more primers that each comprise at least one 2 to 12fold degenerate codon, wherein each primer comprises at least twooligonucleotide sequences that are complementary to a sequence in theparent nucleic acid and code for an amino acid mutation with theexception of cysteine or methionine at one amino acid position encodedby the parent nucleic acid; and (b.) mutating the parent nucleic acid byreplication or polymerase based amplification using the one or moreprimers obtained in (a), wherein replication or amplification of theparent nucleic acid with the one or more primers generates mutatednucleic acids that encode modified antibody variable domains.
 33. Amethod for mutagenesis of an antibody variable domain to obtain modifiedantibody variable domains with mutated amino acid sequences, the methodcomprising: a. identifying one or more amino acid positions in theantibody variable domain for mutagenesis; b. substituting one or more ofthe identified amino acid residues in the antibody variable domain withother amino acid residues excluding cysteine and methionine to generatea library or an array of modified antibody variable domains with mutatedamino acid sequences; c. screening the library or array of modifiedantibody variable domains in an assay for a biological activity of theantibody variable domain; and d. obtaining modified antibody variabledomains having the biological activity of the antibody variable domain.34-36. (canceled)
 37. A method of producing a nucleic acid library withan equal representation of non-redundant amino acid changes at an aminoacid position encoded by a parent nucleic acid encoding an antibodyvariable domain, the method comprising: (a.) providing a set of primersthat each comprise at least one degenerate codon, wherein each primercomprises at least two oligonucleotide sequence that are complementaryto a sequence in the parent nucleic acid and code for an amino acidmutation with the exception of cysteine and methionine at one amino acidposition encoded by the parent nucleic acid, wherein the primers codefor an equal representation of non-redundant amino acid changes at theone position; (b.) hybridizing a primer from the set to the parentnucleic acid; (c.) replicating or amplifying the parent nucleic acidmolecule with the primer to generate nucleic acids that code for aminoacid changes at the one position; (d.) repeating steps (b) and (c) witheach remaining primer from the set; (e.) pooling the nucleic acidsproduced with each primer; and (f.) obtaining a library of nucleic acidsfrom steps (a)-(e) coding for an equal representation of amino acidchanges at the one position.
 38. (canceled)
 39. A method of makingmodified antibody variable domains with mutated amino acid sequences,the method comprising: a. modifying the amino acid sequence of anantibody variable domain to produce amino acid mutations at an aminoacid residue in the antibody variable domain to generate a library or anarray of modified antibody variable domains with mutated amino acidsequences, wherein the amino acid mutations exclude cysteine andmethionine; and b. selecting modified antibody variable domains from thelibrary or the array that have a biological activity of an unmodifiedantibody variable domain. 40-71. (canceled)
 72. A library or an arraycomprising variants of a antibody variable domain sequence, wherein thevariants each comprise an amino acid mutation at one amino acid positionin the sequence of a parent antibody variable domain and wherein theamino acid mutations are not cysteine or methionine. 73-76. (canceled)